Preparation of Template RNA

Successful RT-PCR requires a high quality, intact RNA template. Use the following guidelines to help prepare this template:

• To minimize the activity of RNases that are released during cell lysis, include RNase inhibitors in the lysis mix or use methods that simultaneously disrupt cells and inactivate RNases.

• Take steps to eliminate all potential sources of RNase contamination from glassware, plasticware, reagents, etc.

• Use a product specifically designed for nucleic acid purification to prepare starting template RNA.

• Use purified mRNA as template, rather than total RNA. Starting with poly(A)+ mRNA will greatly increase the likelihood of successful amplification of rare mRNAs, since the proportion of mRNA in a total RNA preparation is quite low (typically, 1–5% of total RNA from a mammalian cell).

• If using mRNA as template, check the integrity of the mRNA by gel electrophoresis before using it in RT-PCR. The mRNA should appear as a smear between approx. 500 bp and 8 kb. Most of the mRNA should be between 1.5 kb and 2 kb.