Reverse transcription of total RNA

The following reverse transcription (RT) protocol is based on several references including:

Sellner and Turbett, Biotechniques 25: 230-34
Sambrook ch 14 pg. 14.20
Gause and Adamovicz "Use of PCR to quantitate relative differences in gene expression" PCR Primer pp 293-311.
Invitrogen protocol for Superscript II

Total RNA is isolated (typically from retinas or whole eyes of juveniles) and quantified using A260/A280 absorption. Based on this, 0.5 ug of total RNA is used in an RT reaction. This RT reaction uses two key enzymes: RNAse inhibitor (Invitrogen RNAse OUT 1077-019) and Superscript III RT (Invitrogen 18080-044). Our previous recipe using cloned RNAse inhibitor (Invitrogen 15518-012) is also given. The Superscript comes with 1st strand buffer and DTT.

Recipe :

Ingredients	RNAseOUT	Old 1	Old 2
Mix together in a 0.5 ml RNAse free tube:
total RNA	0.5 ug = y ul	0.5 ug = y ul	1 ug = x ul
dNTP (0.5 mM each)	1.25 ul of 10 mM each mix	1.25 ul of 10 mM each mix	2.5 ul of 10 mM each mix
poly T primer (50 pmole)	2.5 ul of 10 uM stock	2.5 ul of 10 uM stock	5 ul of 10 uM stock
water as needed	11.875 - y ul	10 - y ul	20 - x ul
total volume	15.625 ul	13.75 ul	27.5 ul
Heat to 65C for 5 min. Quench on ice for 1 min. Then add:
5X 1st strand buffer	5 ul	5 ul	10 ul
0.1 M DTT	2.5 ul	2.5 ul	5 ul
RNase inhibitor	0.625 ul (25U OUT)	2.5 ul (25U cloned)	5 ul (50U cloned)
Quick spin and incubate at RT for 2 min. Then add:
Superscript III	1.25 ul (250U)	1.25 ul (250U)	2.5 ul (500U)
Total volume	25 ul	25 ul	50 ul
Incubate 10 min at room temp. Incubate 50 min at 42C

These steps are usually done by first making a master mix for the dNTP and polyT primer. This is then added to water in each tube prior to adding RNA. Then make a 2nd master mix for 5xbuffer, DTT and RNase inhibitor. It is important that RNAseOUT (or RNAse inhibitor) is always in DTT as it becomes inactivated without DTT. It is convenient to do the incubations in the hybridization oven. Deactivate the reaction at 70C for 15 min (often skip this as first step of qPCR is a 95 C hold for 10 minutes). Store reaction at -20C.

This gives plenty of materials for real time RT PCR. The most RT reaction used in a real time RT PCR reaction is 0.5 ul. So 50 ul of RT reaction is enough for 100 real time reactions. However, 1000x less can be used in a real time reaction. This means there is enough to do an infinite number of real time studies. It is also possible to do half (25 ul) RT reactions which uses less Superscript II.