Quantitating Gene Expression Directly from Cell Lysates Using TaqMan® Real-Time PCR Analysis

• Skip RNA isolation and purification without compromising gene expression results using TaqMan® Gene Expression Assays

• Go from cell culture to real-time PCR in less than 2 hours

• Detect even rare messages without having to isolate RNA

AbstractWe compared expression data for 16 genes in seven cell lines using either DNase-treated cell lysates in Cells-to-cDNA™ II Cell Lysis Buffer (Ambion, an Applied Biosystems Business) or purified RNA isolated using a kit from Manufacturer Q. Samples derived from a broad range of cell concentrations were reverse transcribed using Applied Biosystems High Capacity cDNA Reverse Transcription Kit. cDNA was then amplified using TaqMan® Universal PCR Master Mix and a panel of TaqMan Gene Expression Assays (both from Applied Biosystems). The results were comparable in terms of expression level, linear response to sample dilution, and variance among technical replicates. We also evaluated a smaller set of genes characterized as low expressers, and obtained similar results. Finally we were able to corroborate published observations suggesting that EGFR is highly expressed in HeLa cells and not expressed, or expressed at very low levels, in Raji cells. Ambion’s innovative Cells-to-cDNA II Cell Lysis Buffer provided a simpler, easier alternative to isolating RNA from cultured cells for gene expression analysis. Paired with Applied Biosystems reagents for reverse transcription, TaqMan-based real-time PCR, and Applied Biosystems Real-Time PCR instruments we were able to generate reliable quantitative gene expression data faster and easier than ever before.

Figure 1. RNA Isolation-Free Workflow for Gene Expression Analysis.

1 Cell Lysis and DNase TreatmentCells-to-cDNA™ II Cell Lysis Buffer (P/N AM8723)

DNase I (RNase-free) (P/N AM2222, AM2224)

2

3

Reverse Transcription High Capacity cDNA Reverse Transription Kit(P/N 4368814)

Detection Using Real-Time PCRTaqMan® Universal PCR Master Mix(P/N 4304437)

TaqMan® Gene Expression Assays(P/N 4331182, 4351372)

MSP4 NEW.indd 1 1/23/07 10:48:27 AM

2

IntroductionTraditionally, the first step in gene expression experiments has been to recover pure RNA from experimental samples. Even using the quickest and simplest techniques, however, isolation of RNA requires a significant amount of hands-on manipulation and time. Ambion has developed an innovative reagent, Cells-to-cDNA™ II Cell Lysis Buffer, that lyses cultured mammalian cells while protecting endogenous RNA from degradation. The lysate can serve as an effective template for reverse transcription and Real-Time PCR without having to actually purify the RNA. Cells-to-cDNA II lysates can be efficiently reverse transcribed using Applied Biosystems robust High Capacity cDNA Reverse Transcription Kit. Then, expression levels can be quantitatively detected using Applied Biosystems TaqMan® Universal PCR Master Mix, state-of-the-art TaqMan Gene Expression Assays, and trusted Real-Time PCR Instruments. Furthermore, this protocol can easily be automated for high throughput screening applications.

This RNA isolation-free method for gene expression analysis (see Figure 1) was tested in several cultured mammalian cell lines across a wide range of cell inputs with up to 16 different TaqMan Gene Expression Assays. Cells-to-cDNA II Cell Lysis Buffer affords greatly simplified sample preparation with minimal handling for sequence detection by real-time RT-PCR. The combination of Cells-to-cDNA II lysate prep technology

with the sensitivity and specificity that only TaqMan PCR Chemistry can supply means that, for cultured cells, RNA isolation for gene expression analysis is no longer necessary.

TaqMan® Gene Expression Assays Designed for PCR efficiency, high specificity, and sensitivity using Applied Biosystems powerful bioinformatics pipeline and proprietary software algorithms, TaqMan® Gene Expression Assays use TaqMan probes (5’ nuclease chemistry) that feature a nonfluorescent quencher and incorporate MGB (Minor Groove Binder) technology. There are over 700,000 pre-designed, pre-optimized assays to choose from for human, mouse, rat, Arabidopsis, Drosophila, C. elegans, Rhesus macaque and C. familiaris (dog) genes. In addition, Custom TaqMan Gene Expression Assays are available. Each TaqMan Assay is a ready-to-use, 20X mixture of PCR primers and TaqMan probe designed for amplification using universal cycling conditions so that any combination of assays can be run using the same thermal cycling conditions. Just add TaqMan Universal PCR Master Mix (with or without AmpErase® UNG) and your cDNA sample to generate accurate, reproducible, and truly quantitative gene expression data.

High Capacity cDNA Reverse Transcription Kit Applied Biosystems High Capacity cDNA Reverse Transcription Kit (formerly the High Capacity cDNA Archive Kit) delivers extremely high-quality, single-stranded cDNA from total RNA. It contains all components necessary for the conversion of 0.02–2 μg of total RNA to cDNA in a 20 μL reaction. Reactions are primed with random primers and MultiScribe™ recombinant MuLV reverse transcriptase. The kit has been extensively validated with an array of different RNA templates and quantities, including challenging AU- or GC-rich sequences, and demonstrates consistent, quantitative cDNA synthesis.

TaqMan® Universal PCR Master MixApplied Biosystems TaqMan® Universal PCR Master Mix combines the components needed for the fluorogenic 5’ nuclease PCR technique in one easy-to-use premix. The mix includes carefully optimized proprietary buffer components and stabilizers, as well as AmpliTaq Gold® DNA Polymerase, to enhance reaction performance. TaqMan Universal PCR Master Mix is available in many sizes to fit the needs of your laboratory, and both with and without AmpErase® Uracil-DNA Glycosylase (UNG), which prevents carry-over contamination from previous PCRs.

Figure 2. Experimental Set-up.

Cultured Mammalian Cells

High CapacitycDNA Reverse Transcription Kit

TaqMan® Universal PCR Master Mix

TaqMan® Gene Expression Assays

RNA Isolation Kit (Manufacturer Q)

• Add lysis reagent• Spin through gDNA Column• Add ethanol• Spin through column• Wash column• Wash column • Wash column • Elute RNA

Cell Lysate Workflow

• Add Cells-to-cDNA™ II Lysis Buffer, heat to 75°C• Add DNase I, heat at 37°C • Heat at 75°C to inactivate DNase

8 steps3 steps

MSP4 NEW.indd 2 1/23/07 10:48:29 AM

3

Gene Expression Analysis Results are Comparable Using Either Purified RNA or Cell LysatesFigure 2 outlines the experimental process used to compare gene expression data from purified RNA versus direct real-time RT-PCR of cell lysates prepared using Ambion’s Cells-to-cDNA II Cell Lysis Buffer and DNase I. Cells from the seven cell lines shown in Figure 3 were collected, diluted with culture media to final cell concentrations ranging from 10 to 300,000 cells per lysis, washed with PBS, and lysed in Cells-to-cDNA™ II Cell Lysis Buffer. Lysates were then treated with DNase I. By diluting cells before lysis, the reagent concentrations in all the lysate samples were equivalent, and any cell number effects

on lysis were captured. To obtain purified RNA, cells were processed using an RNA isolation kit from Manufacturer Q according to the manufacturer’s instructions. The purified RNA was then diluted in water to mimic the cell concentrations used in the cell lysate method.

The Cells-to-cDNA II cell lysate method consists of only three pipetting and/or heating steps. Isolating RNA using the kit from Manufacturer Q, requires at least eight steps, not including emptying wash solutions from each collection tube after centrifugation. Sample prep using the cell lysate method is significantly easier, and requires much less manual manipulation than does RNA purification. For experiments

Cell Line Type Source

HeLa adherent Cervical Adenocarcinoma

Raji suspension Burkitt’s Lymphoma

Jurkat suspension T-cell Leukemia

MCF-7 adherent Breast Adenocarcinoma

HEPG2 adherent Liver Carcinoma

293 adherent Embryonic Kidney

U87-MG adherent Neuronal

Figure 3. Cell Lines Used in the Study.

Gene Symbol Gene Expression Assay Gene Name

ACTB Hs99999903_m1 actin-beta

GAPD Hs99999905_m1 glyceraldehyde-3-phosphate dehydrogenase

PPIA Hs99999904_m1 peptidylprolyl isomerase A (cyclophilin A)

B2M Hs99999907_m1 beta-2-microglobulin

HPRT1 Hs99999909_m1 hypoxanthine phosphoribosyltransferase 1

PGK1 Hs99999906_m1 phosphoglycerate kinase 1

RPLP0 Hs99999902_m1 ribosomal protein, large, P0

TBP Hs99999910_m1 TATA box binding protein

TFRC Hs99999911_m1 transferrin receptor

GUSB Hs99999908_m1 glucuronidase, beta

CDKN1A Hs00355782_m1 cyclin-dependent kinase inhibitor 1A

CTNNB1 Hs00170025_m1 catenin (cadherin-associated protein), beta 1

MYC Hs00153408_m1 myelocytomatosis viral oncogene homolog

ENO1 Hs00361415_m1 enolase 1, (alpha)

TP53 Hs00153340_m1 tumor protein p53

MAPK14 Hs00176247_m1 mitogen-activated protein kinase 14

Figure 4. TaqMan® Gene Expression Assays Used in the Initial Study. Assays shown in color target genes characterized as endogenous controls.

MSP4 NEW.indd 3 1/23/07 10:48:29 AM

4

Figure 5. Gene Expression Data is Equivalent Using Cells-to-cDNA™ II Lysates or Purified Total RNA. Representative quantitative gene expression data are shown for 12 Applied Biosystems TaqMan® Gene Expression Assays conducted on 10 increasing quantities of sample. Plots of CT vs. cell number for the indicated TaqMan Assay compare data from the Cells-to-cDNA II cell lysate method to that obtained using purified RNA with the same RT-PCR reagents.

Cell Count

10 30 100

300

1000

3000

1000

0

3000

0

1e+

05

3e+

05

Cell Count

10 30 100

300

1000

3000

1000

0

3000

0

1e+

05

3e+

05

Cell Count

10 30 100

300

1000

3000

1000

0

3000

0

1e+

05

3e+

05

Cell Count

10 30 100

300

1000

3000

1000

0

3000

0

1e+

05

3e+

05

40

35

30

25

20

15

GAPD PPIA HPRT1 B2M

CT

40

35

30

25

20

15

GUSB PGK1 RPLP0 MAPK14

CT

40

35

30

25

20

15

CTNNB1 ENO1 TP53 CDKN1A

CT

LysatesTotal RNA

GAPD

40

35

30

25

20

15

CT

Cell Count

10 30 100

300

1000

3000

1000

0

3000

0

1e+

05

3e+

05

LysatesTotal RNA

MSP4 NEW.indd 4 1/23/07 10:48:31 AM

5

involving many samples this is an advantage for avoiding sample confusion and user fatigue. But even for relatively small experiments, minimizing sample handling and hands-on time is an important benefit of the cell lysate method.

After sample preparation was complete, 10 µL aliquots of each sample were reverse transcribed in 50 µL reactions using Applied Biosystems High Capacity cDNA Reverse Transcription Kit. With its robust capacity, high efficiency, and rigorous linearity, the High Capacity cDNA Reverse Transcription Kit can synthesize maximal yields of cDNA even from the wide range of sample amounts that were used in this study. Finally, 10 µL of each reverse transcription reaction served as template in a real-time PCR using one of 16 TaqMan Gene Expression Assays (Figure 4). Each 100 µL lysate or RNA sample provided template to assay expression levels of eight different genes. There were 4 technical replicates of each TaqMan Assay, with each PCR containing 2% of the initial 100 µL sample. The reactions were run with Applied Biosystems TaqMan® Universal PCR Master Mix on a 7900HT Fast Real-Time PCR System under universal cycling conditions.

Figure 5 shows a representative subset of the gene expression data obtained using either Cells-to-cDNA II lysate or purified RNA isolated from seven cell lines, tested at 10 dilutions. Both sample types show extremely similar trend lines. Although the CT values were often 1–2 CT higher for the lysate samples compared to the purified RNA samples, the slope and linearity of the assay results were comparable.

Furthermore, we obtained good sensitivity and reproducibility across the entire range of cell amounts tested in these experiments, 10 to 300,000 cells per 100 µL lysis reaction. Overall, our analysis determined the following:

• The slopes of CT vs. cell number for lysates are comparable to those from purified RNA. This is an indication that the complex mixture of biomolecules present in cell lysate samples does not impact the RT or PCR efficiency.

• An analysis of the correlation between cell number and CT showed that >95% of our assays have a linear dynamic range which extends to input levels of 30,000 cells per lysis, confirming that residual cellular debris has no detectable influence on RT-PCR linearity up to this concentration.

• Even at 300,000 cells per lysis, which roughly corresponds to the number of cells in 12 confluent wells of a 96-well plate, more than 50% of the TaqMan Assays and cell line combinations we tested maintained full linearity. (It is important to evaluate a range of cell counts to determine the actual dynamic range of your own system.) Figure 6 shows the correlation coefficient (R2) of the TaqMan Assays across all cell lines for cell lysates versus purified RNA.

• Variance among technical replicates for cell lysates and purified RNA was the same.

Figure 6. Linearity is Equivalent using Cells-to-cDNA™ II Lysates or Purified Total RNA. R2 describes the correlation between the measured CT values and the log of the reaction template quantity. It is a statistical estimate of how close to linear the CT measurements are across the number of cells used. This graph shows the density of R2 values compiled from all of the experiments in the study. The pattern from both Cells-to-cDNA II lysates and purified RNA are virtually identical indicating that the gene expression data from lysates maintains linearity.

35

30

25

20

15

10

5

0

0.75 0.80 0.85 0.90 0.95 1.00

Dens

ity

R2

LysatesTotal RNA

MSP4 NEW.indd 5 1/23/07 10:48:32 AM

6

Even for Low Expressing Genes, Conclusions were the Same Using Cell Lysates or Purified RNANext we tested whether the RNA isolation-free method would also work well for expression studies of genes characterized as low expressers. Using the same experimental design and dilution series as in the initial set of experiments (Figure 2), we assayed HeLa and Raji cells for expression of eight genes known to be expressed at relatively low levels (Figure 7). As in the initial study, the results from TaqMan Gene Expression Assays conducted using cDNA generated from Cells-to-cDNA II lysates or purified RNA were in close agreement (Figure 8). Overall, our analysis determined that the CT values, standard deviation among technical replicates, and slopes and linearity of low expresser assay results were no different for the two sample preparation methods. These results indicate that, despite their relatively low concentration in the complex mix of cellular debris in the cell lysates, there is no differential loss of signal for low expressing genes in comparison to purified RNA. Thus, the RNA isolation-free method can be used with these more challenging targets as well as for higher expressing genes.

RNA Isolation-Free Gene Expression Analysis of EGFRIt has been well established that EGFR is highly expressed in many epithelial tumors and solid tumor-derived cell lines such as HeLa (Uyemura 2005). On the contrary, published flow cytometry evidence indicates that EGFR is not expressed in Raji cells which are derived from Burkitt’s lymphoma (Vinante 1999). In addition, Moroni (2001) found that EGFR is not expressed in EB-3 cells which are derived from a different B-cell lymphoma. Our experiments directly compared EGFR expression in HeLa and Raji cells, and our results confirm the published observations. We found significant differential expression of EGFR between HeLa and Raji cells; both Cells-to-cDNA II lysate and purified RNA data show an approximately 10 CT difference in signal (Figure 9).

Figure 9. Differential Expression of EGFR is Detected with Both Cells-to-cDNA™ II Lysate and and Purified Total RNA. CT vs. Cell Number for an EGFR TaqMan® Gene Expression Assay with HeLa or Raji cell lysates or purified RNA show virtially identical results.

Figure 8. Gene Expression Data from Low Expressing Genes is Equivalent using Cells-to-cDNA™ II Lysates or Purified Total RNA. Quantitative gene expression data for six Applied Biosystems TaqMan® Gene Expression Assays conducted on samples from ten increasing quantities of HeLa cells. Plots of CT vs. cell number for the indicated TaqMan Assay compare data from the Cells-to-cDNA II cell lysate method to that obtained using purified RNA with the same RT-PCR reagents.

Gene Symbol Assay Gene Name

CYP2D6 Hs00164385_m1 cytochrome P450, fam. 2, subf. D, polyp. 6

EGFR Hs00193306_m1 epidermal growth factor receptor

FMR1 Hs00233632_m1 fragile X mental retardation 1

IGF2 Hs00171254_m1 insulin-like growth factor 2

MMP9 Hs00234579_m1 matrix metallopeptidase 9

PLAU Hs00170182_m1 plasminogen activator, urokinase

F8 Hs00252034_m1 coagulation factor VIII

MAOA Hs00165140_m1 monoamine oxidase A

Figure 7. TaqMan® Gene Expression Assays for Low Expressing Genes Used in the Study.

FMR1

ERBB4

ACVR1B

LTBP4

CT

CT

CT

Cell Count Cell Count

ACVR1LRRC28

Lysates Total RNA

Lysates Total RNA

Lysates Total RNA

Lysates Total RNA

LysatesTotal RNA

LysatesTotal RNA

40

35

30

25

20

15

10 30 100

300

1000

3000

1000

0

3000

0

1e+

05

3e+

05

40

35

30

25

20

15

10 30 100

300

1000

3000

1000

0

3000

0

1e+

05

3e+

05

40

35

30

25

20

15

10 30 100

300

1000

3000

1000

0

3000

0

1e+

05

3e+

05

40

35

30

25

20

15

10 30 100

300

1000

3000

1000

0

3000

0

1e+

05

3e+

05

40

35

30

25

20

15

10 30 100

300

1000

3000

1000

0

3000

0

1e+

05

3e+

05

40

35

30

25

20

15

10 30 100

300

1000

3000

1000

0

3000

0

1e+

05

3e+

05

Cell Count

10 30 100

300

1000

3000

1000

0

3000

0

1e+

05

3e+

05

40

35

30

25

20

15

C T

HeLa Lysates HeLa Total RNA Raji Lysates Raji Total RNA

EGFR

MSP4 NEW.indd 6 1/23/07 10:48:35 AM

7

ConclusionsGene expression data from the Applied Biosystems RNA isolation-free procedure in which cell lysates are used directly in real-time RT-PCR were comparable to data obtained using total RNA purified with a kit from Manufacturer Q.

In this study we were particularly interested in seeing whether samples prepared in Ambion’s Cells-to-cDNA™ II Cell Lysis Buffer would perform well with Applied Biosystems reverse transcription and TaqMan® Gene Expression Assay reagents compared to samples prepared using traditional RNA purification methods. The results were unequivocal. Not only did the Cells-to-cDNA II lysate samples perform well, the results indicate that with cultured mammalian cells, it is no longer necessary to isolate RNA for gene expression analysis using TaqMan Assays.

Using Cells-to-cDNA II Cell Lysis Buffer, you can omit RNA isolation and go straight into real-time RT-PCR without compromising your experiments. With the RNA isolation-free workflow for gene expression analysis, days spent standing in front of a microcentrifuge, emptying collection tubes, and pipetting wash after wash are no longer necessary. Using this combination of Ambion and Applied Biosystems products, we were able to quickly screen several cell lines across a number of TaqMan Assays. Results were comparable to those using total RNA and good linearity was seen across a cell concentration gradient of 10–300,000 cells per 100 µL lysis. The Cells-to-cDNA II lysis protocol dramatically reduced the number of steps in sample preparation, thus reducing handling time. The Cells-to-cDNA II lysate was similar to purified RNA in reverse transcription efficiency and showed little or no PCR inhibition even at high cell concentrations. When cDNA generated from the lysates was used with TaqMan Gene Expression Assays, excellent precision across technical replicates was seen and highly reliable gene expression data was generated—comparable to that from purified total RNA.

Note: Most of the experiments in this study were conducted using TaqMan® Universal MasterMix. Unpublished results indicate that this protocol will also work well with FAST chemistry and FAST thermal cyling conditions, but we recommend that you test the procedure in your lab before embarking on large experiments.

Scientific ContributorsCatalin Barbacioru

Frances Chan

Laura Chapman

Richard Fekete

David Keys

Annalee Nguyen

Raymond Samaha

ReferencesMoroni M, Veronese S, Schiavo R, Carminati O, Sorensen B, Gambacorta M, and Siena S (2001) Epidermal Growth Factor Receptor Expression and Activation in Nonseminomatous Germ Cell Tumors. Clinical Cancer Research 7: 2770–2775

Uyemura T, Takagi H, Yanagida T, and Sako Y (2005) Single-Molecule Analysis of Epidermal Growth Factor Signaling that Leads to Ultrasensitive Calcium Response. Biophysical Journal 88:3720–3730.

Vinante F, Rigo A, Papini E, Cassatella M, and Pizzolo G (1999) Heparin-Binding Epidermal Growth Factor-Like Growth Factor/Diphtheria Toxin Receptor Expression by Acute Myeloid Leukemia Cells. Blood 93(5):1715–1723.

MSP4 NEW.indd 7 1/23/07 10:48:35 AM

Headquarters 850 Lincoln Centre Drive | Foster City, CA 94404 USA Phone 650.638.5800 | Toll Free 800.327.3002 www.appliedbiosystems.com

International Sales For our office locations please call the division headquarters or refer to our Web site at www.appliedbiosystems.com/about/offices.cfm

For Research Use Only. Not for use in diagnostic procedures.

© Copyright 2007, Applied Biosystems. All rights reserved. Applied Biosystems, AmpErase, and AB (Design) are registered trademarks and MultiScribe is a trademark of Applera Corporation or its subsidiaries in the US and/or certain other countries. Ambion is a registered trademark and Cells-to-cDNA is a trademark of Ambion, Inc. in the U.S. and/or certain other countries. TaqMan and AmpliTaq Gold are registered trademarks of Roche Molecular Systems, Inc. All other trademarks are the sole property of their respective owners.

Printed in the USA, 01/2007 137AP02-01

ORDERING INFORMATION

Description Quantity Part Number

Cells-to-cDNA™ II Cell Lysis Buffer 5 x 10 mL AM8723

DNase I (RNase-free) (2 U/µL) 2000 U AM2222

DNase I (RNase-free) (2 U/µL) 5 x 2000 U AM2224

High Capacity cDNA Reverse Transcription Kit 200 rxns 4368814

TaqMan® Universal PCR Master Mix 200 rxns 4304437

TaqMan® Gene Expression Assays (Inventoried) 250 rxns 4331182

TaqMan® Gene Expression Assays (Non-inventoried) 360 rxns 4351372

RNA Isolation-Free Workflow for Expression Analysis

Green: Ambion kits and reagents. Blue: Applied Biosystems reagents and instruments.

For more information on these products, contact us or visit www.ambion.com (green listings) and www.appliedbiosystems.com (blue listings).

PRODUCTS

• Cells-to-cDNA™ II Cell Lysis Buffer

• DNase I (RNase-free)

1 2 3

PRODUCTS

• High Capacity cDNA Reverse Transcription Kit

PRODUCTS

• TaqMan® Universal PCR Master Mix

• TaqMan® Gene Expression Assays

Cell Lysis and DNase Treatment

Reverse Transcription Detection Using Real-Time PCR

MSP4 NEW.indd 8 1/23/07 10:48:35 AM