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, 1 ug of total RNA is used in an RT reaction. This RT reaction uses two key enzymes: RNAse inhibitor (Invitrogen 15518-012) and Superscript III RT (Invitrogen 18080-044). The Superscript comes with 1st strand buffer and DTT.

A basic RT reaction includes the following:

Mix together in a 0.5 ml RNAse free tube:
1 ug total RNA	x ul
dNTP (0.5 mM each)	2.5 ul of 10 mM each mix
poly T primer (50 pmole)	5 ul of 10 uM stock
water as needed	20 - x ul
total volume	27.5 ul
Heat to 65C for 5 min. Quench on ice for 1 min. Then add:
5X 1st strand buffer	10 ul
0.1 M DTT	5 ul
RNase inhibitor (50 U)	5 ul
Quick spin and incubate at RT for 2 min. Then add:
Superscript II (500 U)	2.5 ul
Total volume	50 ul

Incubate at RT for 10 min. Then incubate at 42C for 50 min. I usually do this in the hybridization oven. Deactivate the reaction at 70C for 15 min. Store reaction at -80 C (-20C is also fine.)

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.