Variation in enzymatic transient gene expression assays

ANALYTICALBIOCHEMISTRY

182,411-418

(1989)

Variation in Enzymatic Transient Gene Expression Assays Tom

Hollon*”

*Department

Received

of

March

and Fayth Microbiology

K. Yoshimuraty2 and TDepartment

of

Biological

Structure,

of

Washington,

Seattle, Washington

98195

16,1989

We examined causes for high variability in data from enzymatic transient gene expression assays. Our results strongly suggest that variation in transfection efficiency is the major cause of data variation and can seriously compromise valid interpretation of data. We compared averaging data from multiple transfections and cotransfection of a second reporter gene as methods for correcting for variation in transfection efficiency. We found that transfection efficiency can be so highly variable that neither method necessarily overcomes the resulting bias in data. Depending upon the degree in variation in transfection efficiency, a combination of the two methods may be advisable. The need to normalize data for transfection efficiency is dependent upon the difference in strengths of promoters being tested and the relative variability of the transfection method used. We also show that the level of reporter gene expression between transfection experiments performed on different days can vary by more than lo-fold. o 1~39 Academic

University

Press, Inc.

Enzyme assays are widely used for the measurement of promoter-directed gene activity of transiently expressed transfected reporter genes. When the level of enzyme activity of the reporter gene product reflects the level of transcription of the reporter gene driven by its promoter, the reporter enzyme assay is a convenient measure of promoter strength. A major advantage of enzymatic transient gene expression assays is that it is generally much easier to measure enzyme activity than mRNA levels. The great usefulness of enzymatic assays has resulted in the development of a variety of reporter genes for enzymatic gene ’ Present address: Institut Paris Cedex 15, France. ’ To whom correspondence

Pasteur, should

25 Rue

du Docteur

be addressed.

0003-2697/89 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

Roux,

75724

expression assays (l-8) and of faster and more sensitive methods of measuring the activity of the most widely used reporter gene, chloramphenicol acetyltransferase (CAT)3 (9-14). Along with development of reporter gene assays has come a growing awareness that transient gene expression assays have limitations and that care must be used both in the design of experiments and in the interpretation of the resulting data. Factors which should be taken into account for these types of experiments include differences in the stability of transfected DNA between cell lines (15); a saturation effect for gene expression with increasing amounts of transfected DNA (16,17); plasmid sequences with negative effects on eukaryotic genes in transient assays (18-20) and that function as cryptic promoters (2,21,22); the observation that stable and transient gene expression assays are not always comparable (23,24); and reports that intragenic sequences of widely used reporter genes such as TK, CAT, and neoR apparently can interact with promoter sequences so that assays with different reporter genes sometimes give different results (23-25). Another limitation for which we have found no satisfactory guidance in the literature is the high variability in measurements of reporter enzyme activity sometimes observed when a reporter gene construct is transfected multiple times. Our interest in this matter arose from our initial inability to reconcile results of transfection experiments measuring CAT activities of mutant enhancers from murine leukemia viruses (MLVs). In those experiments we were comparing measured enhancer activities of CAT plasmid constructs transfected once each. We found the strongest MLV enhancer in one 3 Abbreviations used: CAT, chloramphenicol acetyltransferase; MLV, murine leukemia virus; FBS, fetal bovine serum; LTR, long terminal repeat; CPRG, chlorophenol red @-D-galactopyranoside; ONPG, ortho-nitrophenyl-@-D-galactopyranoside; CV, coefficient of variation; p-gal, 8-galactosidase.

411

412

HOLLON

AND

transfection experiment might be the weakest in a second experiment comparing the same enhancers. When we took one CAT plasmid and tested the replicability of the entire transfection and enzyme assay process by transfecting the plasmid multiple times, we found a high degree of variability in the resulting CAT activities. We were surprised because our reading of the literature introducing these assays did not mention any great variability in assay data (26). Our investigations strongly suggest that the major cause of assay variability is variation in transfection efficiency. This conclusion is not original with us (27). However, to our knowledge, data to support this conclusion, demonstrating the potential degree of data variability due to transfection efficiency and the limitations of methods used to normalize for transfection efficiency, have not been previously published. Our results may be useful to others in assessing their own needs for controls to aid the interpretation of unexpectedly variable reporter gene activity measurements. MATERIALS

AND

METHODS

Cell lines. L691, IL2, SL3, EL4, and S194 are murine leukemia cell lines and were grown in 5% fetal bovine serum (FBS) (Hyclone Laboratories, Logan, UT) and RPM1 1640 (GIBCO Laboratories, Grand Island, NY). NIH3T3 murine fibroblasts were grown in minimal essential media (GIBCO) and 10% FBS. The CAT plasmid constructions Plusmid constructs. M2, MMM, M2PrA, AAA, and PrM were derived from a pUCl&based plasmid containing the CAT gene called pUCCAT. The M2 clone contains the CAT gene driven by the long terminal repeat (LTR) of the MCF13 murine leukemia virus. MMM and M2PrA contain mutant MCF13 LTRs and AAA contains a mutant LTR of the Akv murine retrovirus. PrM contains only the promoter of the MCFl3 LTR. The details of these constructions have been described elsewhere (37). Electroporution. Cell densities for L691 murine T cells were between 5 X lo5 and 1.5 X lo6 cells/ml on the day of transfection. Cells were growing in log phase and were at least 85% viable as determined by trypan blue staining. L691 cells were washed and resuspended in RPM1 without serum at 10’ cells in 0.4 ml/transfection. Ten micrograms of CAT construct plasmid and 10 pg @galactosidase plasmid containing the SV40 early promoter (pCH110) were mixed with cells to be transfected. Plasmids were transfected in supercoiled form and the amount of DNA used was within the linear response range of a titration curve. Plasmid preparations were banded twice on CsCl ultracentrifugation density gradients. Transfections were performed by electroporation essentially as described by Potter et al. (28), except that

YOSHIMURA

a cylindrical electroporation chamber design was used which improved transfection efficiency about sixfold over cuvette chamber designs (38). Electroporated L691 cells were placed in RPM1 plus penicillin-streptomycin plus 5% FBS for 2 days before cell protein extracts were harvested. Electroporations of IL2, SL3, EL4, and S194 cell lines were performed in the same way as for L691 cells. Subconfluent NIH3T3 cells were trypsinized, washed, and resuspended in RPM1 without serum at 3 X lo6 cells in 0.4 ml/transfection. Four micrograms of CAT construct plasmid was used for NIH3T3 cell transfections. An ISCO Model 494 electrophoresis power supply was used for electroporations. For all cell lines the power supply range setting was 2000 V, 90 mA, 150 W and the limit controls were volts = lOO%, watts approximately 3 % , and milliamperes approximately 3%. CAT and /3-galactosidase assays. Protein extract harvests and nonchromatographic CAT assays were performed essentially as described by Sleigh (14), except that protein extracts for CAT assays were heat treated at 65°C for 10 min in the presence of 5 mM EDTA in order to denature cellular enzymes interfering with the CAT assay (11). Protein concentrations of harvested cell extracts were determined using the Bradford assay (29). One hundred micrograms of protein was used for each CAT assay. Each ,&galactosidase assay was performed using 100 pg protein extract in 0.25 M Tris, pH 7.8, plus 10 ~1 50 mM CPRG (Chlorophenol Red @-D-galactopyranoside, Boehringer-Mannheim), 70 ~1 Z buffer (30), and deionized water to a total volume of 100 ~1. Z buffer is 0.06 M Na2HP04. 7Hz0, 0.04 M NaH2P04.H20, 0.01 M KCl, 1 mM P-mercaptoethanol, pH 7.0. CPRG is a 10 times more sensitive substrate than the more commonly used ONPG. The assay was performed at 37°C for 150 min and the resulting /3-galactosidase activity was immediately read at OD 574 nm. RESULTS Measuring variation. As a measure of transient CAT expression assay variability, we used the coefficient of variation (CV), a dimensionless number defined as the standard deviation expressed as a percentage of the mean. By using CVs it is possible to compare the relative variation in data sets with very different means as well as the relative variations in different steps along a series of experimental operations. If our transfection and subsequent enzyme assay procedure had been highly reproducible we would have expected CVs in the range of 510%. In Table 1 the CV values show the variability in transfection and transient gene expression assay of a single CAT plasmid construction, MMM, transfected by electroporation (28) multiple times in five murine cell

VARIATION TABLE Coefficients

Reporter gene

of Variation of Murine

IN

GENE

1

for Multiple Cell Lines

Transfections

Number of transfections

Cell type

Cell line

TRANSIENT

CAT CAT CAT CAT CAT

IL2 SL3 L691 s194 NIH3T3

T cell T cell T cell B cell Fibroblast

63 38 38 71 19

5 5 6 5 5

P-gal P-gal

SL3 NIH3T3

T cell Fibroblast

47 35

4 6

Luciferase Luciferase

EL4 s194

T cell B cell

42 64

8 5

Note. Experiments measuring coefficients of variation of multiple transfections of a CAT reporter gene used the plasmid MMM, in which the CAT gene was driven by a mutant murine leukemia virus LTR (37). Plasmids pCHll0 (4) and pRSVLuc (2) were used for CV measurements with P-galactosidase and luciferase reporter genes, respectively. pCHll0 uses the SV40 early promoter and pRSVLuc uses the Rous sarcoma virus LTR promoter.

lines.

This

deed

not

table easily

indicated reproducible.

that

our Other

procedures

were

investigators

inhave

reported CAT assay data variation of similar magnitude (31). As shown in Table 1, we found that multiple transfections of plasmids with P-galactosidase or luciferase reporter genes also resulted in highly variable data. Variability tests with different promoters driving CAT gene expression, different. plasmid preparation methods, and different electroporation devices showed results similar to those in Table 1 (data not shown). Replicability of enzyme assays. We considered that the

variability

in our

results

might

be due

to variation

in

transfection efficiency. Variable transfection efficiency might make a strong promoter appear weak if its plasmid were poorly transfected and similarly, by exceptionally efficient transfection, might make a weak promoter appear strong. Alternatively, we considered the possibility that either the CAT enzyme assay itself was highly variable or that the Bradford method (29) we used to measure protein concentration in our cellular protein extracts (a preliminary step before performing CAT assays) was the major source of variability in our experiments. Having measured what we call the overall CVs in Table 1, we next measured the CVs of assay replicability, which we call the assay CVs. When we examined the replicability of CAT assays from a single protein extract we found that the CAT assay itself was highly replicable, with an assay CV of 6% (29 assays from one L691 cell protein extract). A similar experiment showed that the Bradford assay for protein

EXPRESSION

413

ASSAYS

concentration measurement was very reliable (assay CV = 7%, 27 assays). We tested the variability in the enzyme assay of a second reporter gene, /3-galactosidase (Escherichia coli 1acZ) (4) and found that assay to be highly replicable (assay CV = 4%, 32 assays). We further found that the level of reporter enzyme activities used in this study was linear over a wide range of amounts of protein extracted from transfected cells (Fig. 1). Thus, overall CVs for the entire transfection and assay procedure shown in Table 1 were as much as 10 times greater than the CVs of CAT assay and protein concentration measurement. This indicated that high variability in the data was not due to poor reproducibility of the assays themselves. Variable transfection efficiency is the major cause of variation in enzyme assay data. Because we did not have a way to compare the number of cells which take up and express reporter genes in different transfections, we examined transfection-induced enzyme assay variability by an indirect approach. We reasoned that if we cotransfected two plasmids carrying different reporter genes, CAT and ,&galactosidase, that for a given cotransfection the proportion of cells taking up each plasmid would probably be almost equal. Therefore, in a series of cotransfections, overall CVs of enzyme data for both reporter genes should be similar if data variation was caused by variable transfection efficiency. Moreover, if the cotransfections were ranked in terms of their CAT assay activity, from minimum CAT activity to maximum, and were similarly ranked for &galactosidase activity levels, then for a given cotransfection CAT and P-galactosidase activities ranks should match. Comparable CVs for the overall transfection and assay process of

60‘

1.5

o^ 8

50

2 p

40

.

CAT

o

R-GAL -1.0

Y CL 30 z 5 8 ; 5

0.5

2.

2 m 8 3 ” m

10 ;;: 0

0.0 0

100

200

300 Protein

400 (pg)

500

600

FIG. 1. Proportionality of reporter enzyme activities to amounts of protein from a single cellular extract of transfected cells. Solid circles show CAT activity for increasing amounts of protein from a single cellular protein extract of L691 T cells transfected with CAT plasmid M2. @-Galactosidase activity for increasing amounts of protein from a single cellular protein extract of NIH3T3 fibroblasts transfected with the P-gal plasmid pCHll0 is shown by open circles. Data for the tables and other figures in this paper are based on CAT and &galactosidase assays where 100 pg protein was used per assay.

414

HOLLON

AND YOSHIMURA

being as great as 0.88 by chance was 0.005 for each data set. 8 Three findings that strongly suggest that variable 0.6 2 transfection efficiency was the major cause of variation 8 in our assay data are (i) CAT, P-galactosidase, and pro0.4 2 c!l tein concentration assays were themselves highly replicable; (ii) overall CVs for the transfection and assay pro0.2 = cesswere similar for CAT and @-galactosidase in all four cotransfection groups (Table 2) and were much greater o+ 0.0 than the CVs for assay replication; (iii) trends and correM2 M2PrA MA I’M lation coefficients indicated that the processes causing FIG. 2. CAT and P-galactosidase assay data from four groups of cohigh to low activity variation were highly related for transfections of CAT plasmid constructs and pCHll0 in L691 murine both reporter genes. We concluded that the B-galactosiT cells. The arrangement of the data is explained in the text. Solid dase activity for each cotransfection could be used as a circles are CAT assays and open circles are @-galactosidase assays. The one-dimensional scatter plot of all @-galactosidase data for this measure of relative transfection efficiency for that coexperiment indicates the range of data in 32 electroporations in L691 transfection. The full range of transfection efficiency cells. Nonchromatographic CAT assay measurements are cpm of r4Cvariation in this experiment is indicated by the one-diacetylated chloramphenicol extracted from CAT assays with ethyl acmensional scatter plot at the right side of Fig. 2, which etate (14). showed the distribution of P-galactosidase data from 32 transfections of pCH110. There was a 19.4-fold range both reporter genes and a high degree of correlation of between the highest and the lowest P-galactosidase activities; the overall CV was 58%. activity ranks would strongly suggest that transfection efficiency is the major source of variation in our tranComparing methods of correcting data for variation sient gene expression assays. caused by transfection efficiency. Using @-galactosidase We chose four CAT constructions for multiple co- activities as relative measures of transfection efficiency, transfections with pCH110, a plasmid with the /I-galacwe examined their usefulness in correcting bias in CAT tosidase gene driven by the SV40 early promoter (4). assay data caused by the transfection process. The need Each CAT plasmid was driven by an enhancer or pro- for this type of correction is exemplified by the M2PrA moter mutant derived from the LTR of either the CAT data in Fig. 2 where four of the eight M2PrA coMCF13 or the Akv MLV. With each CAT plasmid con- transfections had the lowest P-galactosidase activities of struction we did eight cotransfections with pCHll0 in the entire experiment. This suggested that some of the L691 murine T cells. Cotransfections comparing the M2PrA CAT activities might be artificially low because four CAT constructs were performed in the same experiof relatively poor transfection efficiencies. ment. We then observed whether, within each cotransThe effect of adjusting CAT assay values for variafection group (i.e., for one CAT plasmid construct), the tions resulting from differing transfection efficiencies is rank of /I-galactosidase activity of each cotransfection presented in Fig. 3. Each CAT assay measurement in matched the rank for CAT activity. Fig. 2 is presented as uncorrected data (open circles) and The results are shown in Fig. 2 and Table 2. In Fig. 2, corrected by the level of /3-galactosidase activity for the CAT assay measurements from eight cotransfections for same cotransfection (open squares). We observed that each CAT plasmid construct (solid circles) were plotted P-galactosidase corrections of CAT assay data for transwith increasing activity from left to right. For each CAT fection-induced variation could be imperfect. An examassay the value of the corresponding P-galactosidase as- ple of this can be seen in Fig. 2 where, although the Bsay from the same cotransfection also was plotted (open galactosidase values followed the same upward trends circles). Thus, in Fig. 2, while CAT activities were arranged in order of increasing rank from left to right, figalactosidase activities were not. TABLE 2 Figure 2 showed for each cotransfection group, correCVs and Correlation Coefficients of CAT and /3-Galactosidase sponding to each CAT plasmid, that there were similar Cotransfections in L691 Cells trends of CAT and P-galactosidase assays. This result suggested that the processes causing variation in the asPrM M2 M2PrA AAA says of the two reporter geneswere highly related. Correlation coefficients for CAT and /?-galactosidase activi44% CAT CV 30% 42% 44% ties (Pearson’s product-moment correlation coefficient) 61% 34% p-gal CV 40% 63% were greater than 0.88 for each cotransfection group Correlation 0.97 0.92 0.95 0.68 (Table 2). The probability of a correlation coefficient s

40-

0.8

0 0

CAT B-GAL

VARIATION

0 0

n

s

0 &

30-

if 2 E x

20-

z 5

lo-

2 0

IN

0

TRANSIENT

BEFORE AFTER

0 4-Oi

8

.:I O

-i”

oE

On

B

rfb

0

-Q-

OM2

M2PrA

AM

+I-, Phi

FIG. 3. CAT assay data from Fig. 2 before and after normalization for transfection efficiency variation with fl-galactosidase activity measurements. Open circles indicate CAT data before normalization for variable transfection efficiency and the open squares represent the data after normalization. Normalized data have been further corrected by the average P-galactosidase activity for the entire experiment, i.e., OD 574 of 0.21. Horizontal lines indicate data averages. In some columns fewer than eight points appear because of data overlap.

from left to right of the CAT data, there were several places in the M2PrA, AAA, and PrM cotransfection groups where the upward &galactosidase trends were broken. At the points where the trends were broken the ranks of enzyme activities did not match. This meant that a few CAT assay values in Fig. 3 (open squares) were over or undercorrected by ,&galactosidase assay transfection efficiency controls. Only in the M2 cotransfection group was there a perfect match between all CAT and P-galactosidase ranks (the probability of perfect rank matchings from eight cotransfections on a random basis was 2.5 X 10e5). Averaging eight CAT assays without benefit of transfection efficiency control adjustments (Fig. 3, open circles) suggested that promoters M2PrA and AAA were close in strength and that M2 was stronger than either. As indicated earlier in the examination of Fig. 2, some M2PrA CAT data seemed biased too low because of several poorly efficient transfections. After the data was corrected for variation in transfection efficiency by using P-galactosidase activity values (open squares), Fig. 3 suggested that M2PrA was a more powerful promoter than AAA. This demonstrated that simply averaging unnormalized data would not necessarily overcome the bias in the data caused by variable transfection efficiency. Comparison of M2PrA and M2 in Fig. 3 was less conclusive. The comparison of individual data points corrected for transfection efficiency, averaging uncorrected data, or averaging p-galactosidase-normalized CAT assay data was not able to conclusively resolve which promoter was stronger. There was still considerable overlap of data of the two promoters after ,&galactosidase adjustments. Comparing averages of P-galactosidase-adjusted data suggested that M2PrA was 23% more power-

GENE

EXPRESSION

ASSAYS

415

ful than M2, but error calculations of standard errors of the mean suggested that it was possible that the two promoters had equal strength. This illustrated that both averaging and using second reporter gene controls to correct for transfection efficiency had limitations. We note that the overall CVs of the CAT assay data were reduced after normalizing with ,&galactosidase measurements (from 30 to 13%, M2; from 42 to 29%, MrPrA; from 44 to 20%, AAA, and from 44 to 18%, PrM), as would be expected of a control for transfection efficiency. However, in none of these instances was the final P-galactosidase corrected overall CV as small as, for example, the CV (6%) of CAT assay replicability mentioned earlier. It was evident in comparing M2 and PrM in Fig. 3 that the M2 transcription regulation sequences directed much more transcription than those of PrM. This was not surprising given our previous demonstration with stable transfection experiments that M2 has enhancer sequences located in the M2 region deleted to create PrM (32). Judging by the Fig. 3 data for M2 and PrM, M2 would have appeared more powerful than PrM whether they had been compared with one or multiple transfections, with or without transfection efficiency controls. The difference between the strength of M2, with an enhancer, and that of PrM, without one, was too great to be obscured by variable transfection efficiency. But although single transfections of M2 and PrM without applying ,&galactosidase controls might have sufficed to conclude that M2 had more activity than PrM, the range of that difference could have been from 57- to &fold, depending upon which data points in Fig. 3 were used for comparison. After normalization for transfection efficiency variation this range was narrowed (from 32- to U-fold). This indicated that a qualitative demonstration of enhancer activity might not require transfection variation controls, but a more quantitative demonstration may. Our major conclusion from Fig. 3 concerning interpretation of data from enzymatic transient gene expression assays is that if two promoters are close to each other in strength, then the range of their assay data may show considerable overlap. For instance, the data suggest that if AAA, M2, and M2PrA had each been transfected only once without a transfection efficiency control (open circles), it could have been concluded that any of them had the most powerful promoter. Variation in CAT assay replicability would by itself cause some overlap of data of two promoters close in strength. But variation in transfection efficiency greatly increased the range of the assay data beyond what it would otherwise have been. Figure 3, therefore, suggests that variation in transfection efficiency necessitates large differences in promoter strengths to obtain transient assay data that do not overlap.

HOLLON

L:l:

AND

s

A

8

30

2 g d k 5 5 r 5

1

0.1 0.3 0.5 I3 - GAL OD 574

0.7

20

10

0

M2

AAA

M2

AAA

FIG. 4. Variation between experiments. (A) Average M2 CAT activities (not normalized) plotted against average fl-galactosidase activities in cotransfections in L691 cells performed on 4 different days. Each open circle represents data from a different cotransfection experiment. (B) Open bars show the average CAT activities (after fl-galactosidase normalization) of M2 and AAA plasmids cotransfected in L691 cells with pCHll0 in the same experiment. Stippled bars show average normalized M2 and AAA CAT activities in a second experiment with L691 cells on a different day. Error bars are standard errors of the mean. CAT plasmids were cotransfected with pCHll0 six to eight times for all experiments in A and B.

Variation between experiments. For reasons unknown to us, the overall level of transfection efficiency from one experiment to another with the same cell line can be different by a factor of at least 10. The degree of possible variation between experiments is shown in Fig. 4A, in which average M2 CAT activities (not normalized) from six to eight cotransfections with pCHll0 in L691 cells performed on 4 different days were plotted against the average P-galactosidase activities. The range of average M2 CAT activities for the four experiments was from 2.1 X lo3 to 3.15 X lo4 cpm. The OD 574 range of average ,&galactosidase activity was from 0 to 0.6. The data from the experiments suggested that average CAT and fi-galactosidase activities for the four experiments were correlated, with high average activity of one enzyme assay being accompanied by high average activity of the other, and vice versa. We therefore interpreted the variation in these data between experiments to be caused by changes in the overall level of transfection efficiency from one experiment to another. In Fig. 4B, open bars show average M2 and AAA CAT activities (after P-galactosidase normalization) from cotransfections with pCHll0 in the same experiment in L691 cells. Stippled bars show average normalized CAT activities of the same plasmids transfected into L691 cells in a second experiment on a different day. M2 had 2.0 times the CAT activity of AAA in the first experiment and 1.9 times in the second. Although the activities of M2 and AA4 in relation to each other were preserved between the experiments, the absolute levels of CAT activity for both plasmids were higher in the second experiment, demonstrating again variation between experiments.

YOSHIMURA

Assay sensitivity. Figure 4A also shows an effect of practical importance in using cotransfection as a means to normalize data for transfection efficiency, i.e., the relative sensitivity of the enzyme assays in terms of signalto-background. For three of the cotransfection experiments with M2 and pCHll0 in Fig. 4A, we found the CAT assay was from 9 to 17 times as sensitive as the P-galactosidase assay in terms of signal-to-background ratio. For the fourth experiment average M2 CAT activity was 2100 cpm, only 3 times over the background signal, and P-galactosidase activity was at background level. The CAT data thus could not be normalized for the fourth experiment. Because of differences in assay sensitivity and variation in transfection efficiency between experiments, about 15% of our experiments have been discarded because the CAT activity data could not be normalized for transfection efficiency. Part of the difference between CAT and P-galactosidase signal-to-background sensitivity may be due to weak SV40 driven expression of /3-galactosidase in L691 T cells. Stable transfection experiments have indicated that the SV40 early promoter may be weaker in murine T ceil lines than MLV LTRs (32). Another cause for differences in assay sensitivity might be the possible poor context of the translation initiation sequence of the pCHll0 fi-galactosidase (cacAUGa) for efficient translation initiation in eukaryotic cells (33,34). The CAT gene initiator sequence, aaaAUGg, was optimal for efficient translation initiation by the criteria of Kozak (33). The use of a second reporter gene for a transfection efficiency control should take into consideration competition between promoters of cotransfected plasmids for limiting amounts of trans-acting transcription factors, some of which may be shared by different promoters. Scholer and Gruss (16) have shown that promoters of cotransfected plasmids may compete for DNA-binding transcription factors. To lessen the risk of competition, the reporter gene for transfection efficiency control could be transfected in lower molar amounts than the test plasmid. We used a 0.6 pCH110:l.O CAT plasmid molar ratio for cotransfections. Given the lesser sensitivity of the P-galactosidase assay, we could not have further decreased the amount of pCHll0 cotransfected without increasing the number of experiments in which CAT data could not be normalized. Measurement of the amount of reporter plasmid DNA in the nuclei of transfected cells (15) is an alternate method for correction for transfection-induced variation in assay data and avoids the concern about competition. However, in our experience, this was neither as replicable nor as easy a control as a second reporter gene enzyme assay (data not shown).

VARIATION

IN

TRANSIENT

DISCUSSION

We have presented data showing that the amount of variation in enzymatic transient gene expression assays can be very great, with overall CVs exceeding 50%. Our experiments strongly indicate that most of this variation was caused by variable transfection efficiency. The method of averaging unnormalized CAT assay data from as many as eight transfections of each construct proved limited, not always overcoming the bias in data from transfection-induced variation. Cotransfection of a second reporter gene plasmid could be used as a device for correcting enzyme assay data for transfection-induced variation. However, this technique, too, had limitations because CVs of CAT data averaged after ,&galactosidase corrections were still significantly large. The need for transfection variation controls may depend in part on the type of problem investigated. Transfections to compare a promoter with and without an enhancer may result in data so different in magnitude that variable transfection efficiency does not prevent a qualitative demonstration of enhancer activity. However, transfection-induced variation could prevent a quantitative assessment of enhancer strength. On the other hand, if two promoters of nearly equal strength are being tested, assay data variation caused by the transfection process may require much effort to control for variation and demonstrate such equality. In some instances data dependent upon a statistical interpretation may be unavoidable. The need for transfection variation controls may also be dependent upon the degree of variation inherent in the transfection method. It has been suggested (35,36) that manual control of the electrical discharge of an electrophoresis power supply, the electroporation method we used here (28), will give significantly variable discharges and, hence, variable assay data. This criticism may be valid, but as we noted earlier, our tests of one commercial electroporation device (the ProGenetor, Hoefer Scientific) in which discharge was electronically controlled gave us variability measurements similar to those in Table 1 (data not shown). Others have also noted significant discharge variability in commercial electroporation devices (36). Because transfection methods are not usually evaluated in terms of their variability, we are unaware of a transfection method that is highly reproducible in its results. We recommend that investigators assess their own needs for transfection efficiency controls by transfecting a reporter gene plasmid construct 5-10 times in a single experiment and calculating the overall CV of the transfection and assay procedure. This CV would allow the estimation of how wide the range of assay data could be for promoters proportionately stronger and weaker than

GENE

EXPRESSION

417

ASSAYS

that of the promoter used for CV measurement. The need for transfection controls should then be decidable. Currently, our own procedure for measuring gene expression with transient gene expression assays is to perform four cotransfections of each CAT construct plasmid with pCH110. The CAT assay data are corrected for transfection-induced variation with /3-galactosidase measurements. This is then repeated for a second transfection experiment. To correct for variation between experiments we choose a plasmid of high CAT activity to be transfected in all of our experiments. After CAT data of this plasmid has been normalized for transfection efficiency variability within the experiment, its CAT average can serve as a standard for overall transfection efficiency that can be related to other experiments. Data from different experiments can then be combined and averaged, with standard errors of the mean calculated for an indication of uncertainty. Although our experiments have exclusively involved enzymatic gene expression assays, our conclusion that the major contributor of assay variation is the transfection process implies that other measurements for transient gene expression assays, such as mRNA detection, may also need controls for transfection efficiency. ACKNOWLEDGMENTS pCHll0 was a kind gift from Drs. Frank Lee and Gordon Ringold. pRSVLUC was a generous gift from Dr. Donald Helinski. We thank Paul Bornstein for helpful criticism of this manuscript. T.H. was partially supported by a NIH Molecular and Cellular Biology Predoctoral Training grant. This work was supported by Public Health Service Grant CA44166 from the National Institutes of Health to F.K.Y. and a Department of Energy grant (DE-FG 06086 ER60409). During a substantial part of the period that this work was performed F.K.Y. was a Scholar of the Leukemia Society of America.

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