Reverse Transcription Protocol Using SYBR Green Dye Detection

 

Reverse Transcription

Reverse transcription (RT) is the process of converting RNA to cDNA using a reverse transcriptase enzyme and dNTPs. The RT step may be performed on total RNA such that a global cDNA is produced that is representative of all of the RNA transcripts in the sample (usually via a two-step protocol), or in a gene-specific approach such that only the RNA of interest is converted to cDNA (usually following a one-step protocol).

The following experiments can be used as basic RT protocols that can be modified to suit particular requirements. It is customary to either prepare cDNA using a two-step process with subsequent dilution of the cDNA before adding it to the PCR/qPCR, or to prepare a one-step reaction where both processes are performed sequentially.

Equipment

• Quantitative PCR instrument
• Laminar flow hood for RT-qPCR set up (optional)

Reagents

• RNA (stock approximately 1 μg/μL)
• SYBR Green Quantitative RT-PCR kit (Product No. QR0100)
• PCR grade water: PCR grade water (Product No. W1754 or W4502) as 20 mL aliquots; freeze; use a fresh aliquot for each reaction.
• Forward and reverse PCR primers at 10 μM working stocks:
  • Custom oligos can be ordered here. 
    

Instrument	Final Reference Dye Concentration	μL of Reference Dye (per 20 μL Reaction)
Applied Biosystems 5700	1X	0.2
Applied Biosystems 7000	1X	0.2
Applied Biosystems 7300	1X	0.2
Applied Biosystems 7500	0.1X	0.02
Applied Biosystems 7500 Fast	0.1X	0.02
Applied Biosystems 7700	1X	0.2
Applied Biosystems 7900	1X	0.2
Applied Biosystems 7900 HT Fast	1X	0.2
Applied Biosystems 7900HT	1X	0.2
Applied Biosystems StepOnePlus™	1X	0.2
Applied Biosystems StepOne™	1X	0.2
Applied Biosystems ViiA 7	0.1X	0.2
Bio-Rad CFX384™	not used	-
Bio-Rad CFX96™	not used	-
Bio-Rad MiniOpticon™	not used	-
Bio-Rad/MJ Chromo4™	not used	-
Bio-Rad/MJ Opticon 2	not used	-
Bio-Rad/MJ Opticon™	not used	-
Cepheid SmartCycler®	not used	-
Eppendorf Mastercycler® ep realplex	not used	-
Eppendorf Mastercycler® ep realplex2 s	not used	-
Illumina Eco qPCR	not used	-
Qiagen/Corbett Rotor-Gene® 3000	not used	-
Qiagen/Corbett Rotor-Gene® 6000	not used	-
Qiagen/Corbett Rotor-Gene® Q	not used	-
Roche LightCycler® 480	not used	-
Stratagene Mx3000P®	0.1X	0.02
Stratagene Mx3005P™	0.1X	0.02
Stratagene Mx4000™	0.1X	0.02

Supplies

• Sterile filter pipette tips
• Sterile 1.5 mL screw-top microcentrifuge tubes (Product No. CLS430909)
  
  • PCR tubes and plates, select one to match desired format:
  • Individual thin-walled 200 μL PCR tubes (Product No. Z374873 or P3114)
  • Plates
      - 96-well plates (Product No. Z374903)
      - 384-well plates (Product No. Z374911)
    
  • Plate seals
      - ThermalSeal RTS™ Sealing Films (Product No. Z734438)
      - ThermalSeal RT2RR™ Film (Product No. Z722553)

Method

In the example given below, the primer concentrations can be adjusted according to the results of optimization procedures (Primer Concentration Optimization, Primer Optimization Using Temperature Gradient and Assay Optimization and Validation).

1. Place kit components and RNA samples on ice.
2. Mix and then centrifuge briefly to collect contents at the bottom of the tube.
3. Prepare a master mix for each reaction and control requiring RT enzyme plus 10% extra to allow for pipetting    error
          according to Table P11-28.
4. Prepare a master mix for each control requiring NO RT enzyme plus 10% extra to allow for pipetting error referring to
          Table P11-28 but replacing the enzyme with PCR grade water.

Table P11-28. Reaction Master Mix for One-step SYBR Green I RT-PCR.

Reagents	Volume (μL) per Single 25 μL Reaction
2× SYBR Green Quantitative RT-PCR Buffer (Product QR0100)	12.5
Reference dye (optional) Instrument-specific, see Table P4-7	0.025
Primer F (10 μM)	1.125
Primer R (10 μM)	1.125
PCR grade water	9.1
MMLV RT enzyme	0.125

5. Add 1 μL total RNA (250–2500 ng total per reaction) to each PCR tube. If using a PCR plate, follow a plate schematic to ensure that the reaction mix, samples and controls are added to the correct wells.
   
6. Add 24 μL master mix to each well, adding the No RT mix to the minus RT control samples.
   
7. After sealing each reaction or the plate, vortex gently to mix contents.
   
8. Centrifuge briefly to collect components at the bottom of the reaction tubes.
   
9. Set the real-time qPCR according to Table P11-29.

Table P11-29. RT PCR Cycling Parameters for One-step SYBR Green I RT-qPCR.

Cycling Conditions	Temp (°C)	Time
First strand synthesis	42–44	30 min
Denaturation/RT inactivation	94	30 sec
Steps 1–3 are repeated through 40 cycles
Step 1	95	5 sec
Step 2	55	15 sec
Step 3	72	10 sec

10. Run post-reaction melt analysis.