PRIMARY REVIEWER:
Erik Snesrud, Jeremy Hasseman
1. PURPOSE
This protocol describes how to perform real-time Reverse Transcription PCR in a two stepprocedure (Reverse Transcription being a separate step from the PCR) using SYBR Green as the detected fluorophore and ROX as the passive reference.
2. SCOPE
This procedural format is utilized by the Human Colon Cancer and Mouse, Rat, and Arabadopsis microarray projects under the supervision of John Quackenbush and Norman Lee within the Mammalian Genomics Department.
3. MATERIALS
3.1 TaqMan Reverse Transcription Reagents (Applied Biosystems; Cat # N808-0234)
3.2 SYBR Green PCR Master Mix (Applied Biosystems; Cat # 4309155) OR
3.3 QuantiTect SYBR Green PCR Kit (Qiagen; Cat # 204143) (preferred)
3.4 RNaseZAP (Ambion; Cat # 9780)
3.5 MicroAmp Optical 96-well Reaction Plate (Applied Biosystems; Cat # N801-0560) OR
3.6 Optical 384-well Reaction Plate (Applied Biosystems; Cat # 4309849)
3.7 ICycler iQ PCR Plates, 96 well (BioRad; Cat # 2239441) OR
3.8 Thin Wall Tubes; 200 ml (BioRad; Cat # 2239473)
3.9 Optical Caps (8 Caps/Strip) (Applied Biosystems; Cat # 4323032) OR
3.10 Optical Adhesive Covers (Applied Biosystems; Cat # 4311971)
3.11 RNAse and DNAse Free Water
Note: Reaction vessels MUST be DNAse and RNAse free.
4. PREPARATION
4.1 Decontamination
4.1.1 The workbench along with the tube racks and pipetmen should all be sprayed down with RNaseZAP (or any RNAse decontaminant) before use.
4.1.2 RNases are very difficult to kill and can seriously affect the run.
4.2 RNA
4.2.1 RNA should be thawed and stored on ice throughout the reverse transcription preparation and frozen immediately after using.
4.2.2 Repeated freeze/thaw of RNA samples causes significant degradation in RNA integrity.
5. PROCEDURE
5.1 Reverse Transcription (RT)
5.1.1 Thaw all reagents and store on ice.
5.1.2 Maintain an RNAse and DNAse free work environment.
5.1.3 Mix all individual reagents thoroughly and spin down.
Note: A 100-ml RT reaction efficiently converts a maximum of 2.0 mg total RNA to cDNA. Multiple RT reactions should be performed if more than 2.0 mg total RNA is used.
Note: The reverse transcription can be performed using 2 mg in a 100 ml reaction OR using 1 mg in a 50 ml reaction volume.
5.1.4 Master mix (for a single 100 ml reaction):
Note: As with all enzymatic reactions, mix all nonenzymatic components first and then add the enzymatic components.
