Difference between revisions of "20.109(S10):RNA to DNA by RT-PCR (Day5)"

Revision as of 19:38, 28 January 2010

Introduction

The amount of RNA recovered after a column purification is quite small. Thus, in the next step of SELEX, you must increase this small amount of RNA in a stable way. First (today) you convert it to DNA and amplify it, using RT-PCR. Then (next time), you will perform a second in vitro transcription reaction to make more of the RNA aptamer. For this process to work, the 6-5 and 8-12 aptamers must amplify at the same rate, a fact that has been proven for these particular aptamers under the conditions that we are using.

The RT in RT-PCR here stands for reverse transcription, that is, making DNA from an RNA template. We will use a 1-step RT-PCR kit from Qiagen, though the two procedures (reverse transcription and PCR) can be performed separately. The Qiagen kit utilizes a cocktail of two different RT enzymes: Omniscript and Sensiscript, the latter optimized to detect very low abundance sequences. After reverse transcription, these enzymes are inactivated, and a heat-sensitive polymerase is activated so PCR can begin. In sum, the reactions will run for two hours, and we'll spend this time meeting with the writing across the curriculum faculty.

Room in this intro to preview how the binding assay works, or discuss other sundry topics of interest such as the role of natural aptamers in vivo, why heme signaling is interesting, uses of aptamers in therapeutics, use w/ribozymes, etc.

Protocols

Part 1: Recover RNA

1. Centrifuge your samples at max speed for 15 minutes.
   • You and partner can balance your tubes against each other.
2. Collect the supernatants into a temporary waste bottle, such as a 15 mL conical tube.
3. Add one mL of 70% ethanol to each sample, and vortex vigorously.
4. Spin for two minutes at max speed.
5. Repeat the wash step with 1 mL of fresh 70% ethanol.
6. Dry your samples in the fume hood for 10 minutes.
7. Resuspend each sample in 44 μL of RNase-free water by the following procedure:
   • Add the water to the sample, cap tightly, and vortex for 10-20 sec.
   • Quick-spin the samples in the microfuge.
   • Repeat the vortex and the spin steps one more time.

Part 2: RT-PCR

HAVE THEM DO THE CONTROL I DID, OF MAKING SURE 6-5 AND 8-12 AMPLIFY AT SAME RATE?

1. The thermal cycler will be preheated to 50 °C while you prepare your samples. This is required for the procedure to work optimally.
2. Set up your reactions on a cold block as usual. From one of the shared stocks, pipet 30 μL of Master Mix into each well-labeled PCR tubes.
   • In the interests of economy, we will not run control reactions this time. However, if you wanted to test for contamination of your reaction, what component would you leave out?
3. Add 20 μL of your recovered RNA to the Master Mix.
4. The following thermal cycler program will be used:

Part 3: WAC Session

Need to have both Atissa and Neal/Linda come today, so may need to just have teaching faculty do part 4.

Part 4:Run RT-PCR products on gel

After the RT-PCR is completed, students will prepare aliquots and load them onto gels... the teaching faculty will hang around till the gels are done running and post the images online, plus store the sample remainders in the freezer until next time.

For next time

• If you are not presenting for journal club next time, you should work on Part III of the computational assignment, and hand in a picture of the full-length 8-12 sequence with the requested highlighting.
• If you are presenting for journal club, you may hand in the same assignment next week (on Day 7) instead.

Reagent list