cDNA Synthesis Directly from Cells Using SuperScript III CellsDirect cDNA Synthesis System

System Overview

The SuperScript™ III CellsDirect™ cDNA Synthesis System is an optimized kit for synthesizing first-strand cDNA directly from mammalian cell lysate without first isolating the RNA. Lysis and reverse transcription are performed in the same tube, and the resulting first-strand cDNA is ready to use in cloning and PCR. For real-time quantitative RT-PCR, see the note below.
 
In traditional RT-PCR, RNA is first isolated from cells in a time-consuming procedure that can lead to a loss of material. Using the SuperScript™ III CellsDirect™ cDNA Synthesis System, the cells are lysed and the cDNA is generated from the lysate in a single tube with minimal handling and no sample loss. DNase I is added to eliminate genomic DNA prior to first-strand synthesis.
 
This kit has been optimized for small cell samples, ranging from 10,000 cells down to a single cell (as measured by serial dilution). The use of SuperScript™ III Reverse Transcriptase ensures high specificity and high yields of cDNA from small amounts of starting material—as little as 10 pg total RNA.
 
After synthesis, the first-strand cDNA can be amplified with specific primers by PCR without intermediate organic extractions or ethanol precipitations.
 
The diagram below outlines the procedure:







For real-time quantitative RT-PCR (qRT-PCR) from cell lysate, we recommend the SuperScript™ III Platinum® CellsDirect ™Two-Step qRT-PCR Kit (Catalog nos. 11737-030 and 11737-038) or the SuperScript™ III Platinum® CellsDirect™ Two-Step qRT-PCR Kit with SYBR® Green (Catalog nos. 11738-060 and 11738-068). These kits include reagents and protocols that have been specifically optimized for real-time qRT-PCR.
 
Advantages of the Kit

This kit has the following advantages: 

• Compatible with a wide range of mammalian cell types grown under different treatment conditions
• Single-tube format minimizes reagent loss, sample loss, and handling time
• Total lysate volume is used in first-strand cDNA synthesis reaction, providing greater yields with a limited number of cells and allowing for detection of rare transcripts
• SuperScript™ III Reverse Transcriptase, with reduced RNase H activity and higher thermal stability, produces high yields of cDNA in the first-strand synthesis reaction, for greater sensitivity and enhanced detection of rare transcripts
• Generates high-quality cDNA for use in a variety of applications, including cloning and PCR
• Simple protocol takes less than 2 hours

SuperScript™ III RT

SuperScript™ III Reverse Transcriptase is an engineered version of M-MLV RT with reduced RNase H activity and increased thermal stability. The enzyme can be used to synthesize first-strand cDNA at temperatures up to 55° C, providing increased specificity, higher yields of cDNA, and more full-length product than other reverse transcriptases.
 
Because SuperScript™ III RT is not inhibited significantly by ribosomal and transfer RNA, it can effectively synthesize first-strand cDNA directly from total RNA. The concentration of SuperScript™ III RT in this system has been optimized to synthesize first-strand cDNA from total RNA in cell lysate.
 
Control RNA and Primers

The control RNA provided with this system consists of HeLa Total RNA (10 ng/µl). The Forward Control Primer and Reverse Control Primer provided with this kit are designed from the human GAPDH gene and produce a 1.18 kb PCR product.

Introduction

In this step, you lyse your cells in Resuspension Buffer or a Resuspension Buffer/Lysis Enhancer solution and perform a DNase I digestion to remove genomic DNA from the sample.
 
Cell Types and Density

This kit has been optimized for small cell samples, ranging from 1 to 10,000 cells. The performance of this kit was verified using several different mammalian cell lines, including HeLa, COS-7, 293, Jurkat, CV1, and K562. Cells may be grown under variety of conditions and treatments. Any type of culture vessel can be used.
 
We recommend using a maximum of 10,000 cells per reaction. Higher numbers of cells may inhibit reverse transcription and result in reduced yields and/or truncated cDNA product. Make sure that all solutions and equipment that come in contact with the cells are sterile. Always use proper sterile technique and work in a laminar flow hood when handling cells.
 
Required Materials

The following materials are provided by the user:

• Lysis Enhancer (optional) (Catalog no. 11739-010)
• Mammalian cell cultures in growth media
• Coulter Counter or hemacytometer
• Centrifuge (for pelleting cells)
• Incubator, water bath, or thermal cycler preheated to 75°C
• Trypsin (for adherent cell cultures only)
• 1X cold phosphate-buffered saline (PBS), without Ca++ or Mg++
• 0.2-ml thin-walled PCR tubes or 96-well PCR plates
• Ice
• Pipettes

• Resuspension Buffer
• RNaseOUT™ (40 U/µl
• DNase I, Amplification Grade (1 U/µl)
• 10X DNase I Buffer
• EDTA, 25 mM
• Optional: Control HeLa Total RNA

1. Add enough trypsin to cover the adherent cells in your tissue culture dish, plate, or flask (e.g., for a 10-cm dish, use ~1 ml; for a T75 flask, use ~3 ml).
2. Incubate for 5 minutes at room temperate or in a 37°C incubator.
3. Check for cell detachment under a microscope. If cells have not detached, gently tap the disk or flask to dislodge the cells, or let the cells incubate longer, checking them every minute under a microscope.
4. When all the cells have detached, add serum-containing media to a final volume of 10 ml (for 6- and 12-well plates, add a 1X–2X volume of media). Note that the media must contain serum to inactivate the trypsin.
5. Pipet the cells gently up and down to mix, and then transfer the cell suspension to a centrifuge tube.
6. Spin the cells at 200 x g for 5 minutes to pellet.
7. Aspirate the media and wash the cell pellet with 5–10 ml of 1X cold PBS.
8. Spin the cells at 200 x g for 5 minutes to pellet.
9. Aspirate the PBS and resuspend the pellet in 500 µl to 1 ml of 1X cold PBS. Mix the cell solution gently.
10. Collect a small aliquot to verify that the cells are at the desired concentration. Determine cell density electronically using a Coulter Counter or manually using a hemacytometer chamber.
11. Adjust the cell density using cold PBS so that it falls within the range of 1–10,000 cells/µl. Count the cells again to verify cell concentration.
12. To a 0.2-ml thin-walled PCR tube or plate well on ice, add 10 µl of Resuspension Buffer. Then add 1 µl of Lysis Enhancer or 1 µl of RNaseOUT™ (40 U/µl).
13. Transfer 1–2 µl of cells (<10,000 cells) to the PCR tube/well.
    

Control: For the control reaction, add 1 µl of the Control HeLa Total RNA to the PCR tube or plate well instead of cell lysate.


14. Transfer the tube/plate to an incubator, water bath, or thermal cycler preheated to 75°C and incubate for 10 minutes.

Control: For the control reaction, incubate for 3 minutes.


15. After incubation, spin briefly to collect the condensation and proceed to DNase I Digestion.

Higher volumes may be required. Cells should be completely covered by solution.
 
Extra Resuspension Buffer may be required for lysing cells in larger-volume tissue-culture wells. Extra Resuspension Buffer is provided in catalog no. 11739-010, which includes 10 ml of Resuspension Buffer and 1 ml of Lysis Enhancer.

 
Lysing Cells in Tissue-Culture Wells

Note:   Seed cells in tissue-culture wells so that 10 µl of resuspended cells will yield the desired concentration.
 
For adherent cells grown in tissue-culture wells, perform the following lysis procedure.
 

1. Aspirate the media in each well and wash each well with 1X cold PBS. Aspirate the PBS. 
2. Add Resuspension Buffer or Resuspension Buffer/Lysis Enhancer solution (recommended; see page 4) to each well. See the table titled Amount of Resuspension Buffer in Tissue-Culture Wells on page 5 for amounts. The buffer should cover the cells in the well.
3. Incubate the plates on ice for up to 10 minutes. During that period, tap the plate periodically and check the cells under a microscope every 2–3 minutes to see whether they have detached or burst.
4. After 10 minutes, gently pipet the cells up and down to dislodge the remaining attached cells. If the cells are difficult to detach, incubate the plates at room temperature or 37°C for an additional 5 minutes, checking the cells under a microscope periodically.
5. Count the cells or estimate their density based on the seeding density (10 µl should contain <10,000 cells).
6. Transfer 10 µl of the cell suspension to a 0.2-ml thin-walled PCR tube or plate well.
7. Control: For the control reaction, add 10 µl of Resuspension Buffer to a PCR tube or plate well, and then add 1 µl of Control HeLa Total RNA.
8. Add 1 µl of RNaseOUT™ (40 U/µl) to the PCR tube/well.
9. Transfer the tube/plate to an incubator or thermal cycler preheated to 75 °C and incubate for 10 minutes.
10. Control: For the control reaction, incubate for 3 minutes.
11. After incubation, spin briefly to collect the condensation, and proceed to DNase I Digestion.

In this step, you treat the cell lysate with DNase I to degrade any contaminating DNA.
 

  1.     Place each tube/plate from Step 15 of Lysing Adherent Cells or Cells in Suspension, or Step 9 of Lysing Cells in Tissue-Culture Wells, (see Lysing Cells) on  ice, and add the following:

  3.     Incubate for 5 minutes at room temperature. 

 Note:   A longer incubation time (up to 10 minutes) may be used for larger samples (>1,000 cells). However, incubation times exceeding 10 minutes can greatly reduce cDNA yield.

  4.     Spin briefly, and add 1.2 µl of 25 mM EDTA to each tube/well on ice. Mix by gently pipetting up and down, and spin briefly to collect the contents.

  5.     Incubate at 70°C for 5 minutes.

  6.     Spin briefly and proceed to First-Strand cDNA Synthesis.

Required Materials
 
The following materials are provided by the user:

• Thermal cycler preheated to 70°C
• Ice
• Pipettes

• Oligo(dT)20 (50 µM)
• 10 mM dNTP Mix)
• 5X RT Buffer)
• RNaseOUT™ (40 U/µl))
• SuperScript™ III RT (200 U/µl))
• 0.1 M DTT)
• RNase H (2 U/µl)

  2.     Mix by gently pipetting up and down, and spin the tube briefly to collect the contents.
 
  3.     Incubate the tube at 70°C for 5 minutes. Spin the tube briefly to collect the condensation.
 
  4.     Place the tube on ice for 2 minutes, and then add the following: 

  5.     Mix by gently pipetting up and down, and spin the tube briefly to collect the contents.
 
  6.     Transfer the tube to a thermal cycler preheated to 50°C. Incubate for 50 minutes.
 
  7.     Inactivate the reaction at 85°C for 5 minutes.
 
  8.     Add 1 µl of RNase H (2 U/µl) to each tube and incubate at 37°C for 20 minutes.

Note:   This step is optional if you are amplifying short targets (<1.0 kb) in end-point PCR.
 
  9.     Chill the reaction on ice.
 
10.     Store the single-stranded cDNA at –20°C, or proceed directly to PCR amplification.

Introduction

The first-strand cDNA generated using this kit can be used directly in PCR without additional purification. This section provides example protocols for PCR and high-fidelity PCR.

 
PCR Enzymes

For amplifying the first-strand cDNA generated using this kit, we recommend Platinum^® Taq  DNA Polymerase for targets < 1.0 kb and Platinum^® Taq  DNA Polymerase High Fidelity for targets > 1.0 kb.
 
Platinum^® Taq DNA Polymerase is recombinant Taq  DNA polymerase complexed with proprietary Platinum^® antibodies that block polymerase activity at ambient temperatures. Activity is restored after the denaturation step in PCR cycling at 94°C, providing an automatic “hot start” for Taq DNA polymerase in PCR. Hot starts in PCR provide increased sensitivity, specificity, and yield, while allowing assembly of reactions at room temperature. The use of Platinum^® antibodies helps reduce PCR optimization requirements, reaction set-up and handling time, and contamination risk, thereby improving PCR results for templates up to 5 kb.
 
Platinum^® Taq DNA Polymerase High Fidelity is a mixture of recombinant Taq DNA polymerase, Pyrococcus species GB-D polymerase, and Platinum^® Taq antibody. Platinum^® antibody complexes with Taq  DNA polymerase and inhibits activity at ambient temperatures, allowing room-temperature setup. Activity is restored after the PCR denaturation step at 94°C, providing an automatic “hot start” for the enzyme and increasing specificity, sensitivity, and yield.
 
Pyrococcus species GB-D polymerase is a proofreading enzyme that possesses a 3’ to 5’ exonuclease activity. The enzyme mixture in Platinum^® Taq  DNA Polymerase High Fidelity results in a six-fold increase in fidelity over Taq  DNA polymerase alone and allows amplification of simple and complex DNA templates over a large range of target sizes, up to 12 kb with no optimization.

• Since PCR is a powerful technique capable of amplifying trace amounts of DNA, take all appropriate precautions to avoid sample contamination.
• Annealing and extension conditions are dependent on primer Tm, and should be determined independently for each reaction.
• If PCR efficiency is not optimal, repeat the reaction with a primer titration from 100 to 500 nM (final conc.) in 100 nM increments.

1. Add the following components to a sterile 0.2- or 0.5-ml PCR tube or plate well at room temperature or on ice. For multiple reactions, prepare a master mix of common components.



Components	Volume	Final Conc.
10X PCR Buffer, Minus Mg	5 µl	1X
10 mM dNTP mixture	1 µl	0.2 mM each
50-mM MgCl2	1.5 µl	1.5 mM
Sense primer (10 µM)	1 µl	0.2 µM
Antisense primer (10 µM)	1 µl	0.2 µM
cDNA from Step 10	2 µl	--
Platinum® Taq DNA Polymerase	0.4 µl	2.0 units*
Autoclaved, distilled water	to 50 µl	n/a


 

2. Mix contents of the tubes and overlay with 50 µl of mineral or silicone oil, if necessary.


3. Cap the tubes and centrifuge briefly to collect the contents.


4. Incubate tubes in a thermal cycler at 94°C for 30 seconds to 2 minutes to denature the template and activate the enzyme.


5. Perform 30–40 cycles of PCR amplification as follows:
   
   Denature      94°C for 15–30 seconds
   Anneal         55–65°C for 30 seconds
   Extend         72°C for 1 minute per kb


6. Maintain the reaction at 4°C after cycling.  The samples can be stored at -20°C until use.


7. Analyze the products by agarose gel electrophoresis and visualize by ethidium bromide staining. Use appropriate molecular weight standards.

1. Add the following components to a sterile 0.2- or 0.5-ml PCR tube or plate well at room temperature or on ice. For multiple reactions, prepare a master mix of common components.




Components	Volume	Final Conc.
10X High Fidelity PCR Buffer	5 µl	1X
10 mM dNTP mixture	1 µl	0.2 mM each
50-mM MgSO4	2 µl	2 mM
Sense primer (10 µM)	1 µl	0.2 µM
Antisense primer (10 µM)	1 µl	0.2 µM
cDNA from Step 10	>1 µl	--
Platinum® Taq DNA  Fidelity	0.2 µl	1.0 unit*
Autoclaved, distilled water	to 50 µl	n/a


   *1.0 unit is sufficient for amplifying most targets. In some cases, more enzyme may be required (up to 2.5 units).
 

2. Mix contents of the tubes and overlay with 50 µl of mineral or silicone oil, if necessary.


3. Cap the tubes and centrifuge briefly to collect the contents.


4. Incubate tubes in a thermal cycler at 94°C for 30 seconds to 2 minutes to denature the template and activate the enzyme.


5. Perform 30–40 cycles of PCR amplification as follows:
   
   Denature      94°C for 15–30 seconds
   Anneal         55–65°C for 30 seconds
   Extend         68°C for 1 minute per kb


6. Maintain the reaction at 4°C after cycling.  The samples can be stored at -20°C until use.


7. Analyze the products by agarose gel electrophoresis and visualize by ethidium bromide staining. Use appropriate molecular weight standards.

1. Prepare a PCR mixture for each control reaction from Step 10. For each control reaction, add the following to a sterile 0.2- or 0.5-ml PCR tube or plate well at either room temperature or on ice: 




Components	Volume
DEPC-treated water	38.1 µl
10X PCR buffer minus Mg++	5 µl
50 mM MgCl2	1.5 µl
10 mM dNTP mix	1 µl
Forward Control Primer (10 µM)	1 µl
Reverse Control Primer (10 µM)	1 µl
cDNA from control RNA/ negative RT control, Step 10	2 µl
Platinum® Taq  DNA polymerase (5 units/µl)	0.4 µl
Final Volume	50 µl


 

2. Mix the contents of the tube. Centrifuge briefly to collect the reaction components.


3. Place reaction mixture in preheated (94°C) thermal cycler. Perform an initial denaturation step: 94°C for 2 min.


4. Perform 40 cycles of PCR:
   
   Denature      94°C for 15 seconds
   Anneal          60°C for 30 seconds
   Extend          72°C for 1 minute
   
     Note:  For slow-ramping thermal cyclers, follow manufacturer’s directions.


5. Upon completion, maintain reactions at 4°C.


6. Analyze 10 µl of each sample using agarose gel electrophoresis and ethidium bromide staining. A 1.18-kb band corresponding to at least 25 ng of product should be visible for the control sample. No band should be visible for the negative RT control sample.

1. Berger, S.L. and Kimmel, A.R. (1987) Methods Enzymol 152, 316.
2. Bracete, A.M., Mertz, L.M., Fox, D.K. (1999) Focus® 21, 38.
3. Chomczynski, P. (1993) Biotechniques Vol. 15, 532.
4. Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156.
5. Compton, T. (1990) in PCR Protocols: A Guide to Methods and Applications Innis,  M., Gelfand, D., Sninsky, J., and White, T., eds.), p. 39, Academic Press, Inc.
6. D’Alessio, J. M., Gruber, C. E., Cain, C., and Noon, M. C. (1990) Focus® 12, 47. 
7. Frohman, M.A., Dush, M.K, and Martin, G.R. (1988) Proc. Nat. Acad. Sci USA 85, 8998.
8. Gerard, G.F. (1994) Focus® 16, 102.
9. Gerard, G.F., D’Alessio, J.M., and Kotewicz, M.L. (1989) Focus® 11, 66.
10. Gerard, G.F., Schmidt, B.J., Kotewicz, M.L., and Campbell, J.H. (1992) Focus® 14, 91.
11. Hu, A.W., D'Alessio, J.M., Gerard, G.F., and Kullman, J. (1991) Focus® 13, 26.
12. Lee, C.C. and Caskey, T. (1990) in PCR Protocols: A Guide to Methods and Applications (Innis, M., Gelfand, D., Sninsky, J., and White, T., eds.), p. 46, Academic Press, Inc.
13. Sambrook J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
14. Simms, D., Cizdziel, P.E., and Chomczynski, P. (1993) Focus® 15, 99.
15. Westfall, B., Sitaraman, K., Solus, J., Hughes, J., and Rashtchian, A. (1997) Focus® 19, 46.
16. Westfall, B., Sitaraman, K., Berninger, M., and Mertz, L.M. (1995) Focus® 17, 62.
17. Westfall, B., Sitaraman, K., Lee, J., Borman, J. and Rashtchian, A. (1999) Focus® 21, 49.
18. Takagi, M., Nishioka, M., Kakihara, H., Kitabayashi, M., Inoue, H., Kawakami, B., Oka, M., and Imanaka, T. (1997) Appl. Environ. Microbiol. 63, 4504.
19. Sitaraman, K., Darfler, M., and Westfall, B. (1999) Focus® 21, 10.
20. Nathan, M., Mertz, L., Fox, D. (1995) Focus® 17, 78.
21. Schwabe, W., Lee, J.E., Nathan, M., Xu, R.H., Sitaraman, K., Smith, M., Potter, R.J., Rosenthal, K., Rashtchian, A., Gerard, G.F. (1998) Focus® 20, 30