General Materials

• Primers (see below for design)
• ABI high capacity reverse transcription kit with random primers (from LifeTech freezer in MBB supply room)
• RNase/DNase free water
• Sybrgreen 2x PCR master mix (from LifeTech freezer in MBB supply room)
• ABI 384/96 well optical plates (ThermoFisher cat#4309849/cat#N8010560)
• Thermaseal Transparent sealing films for Real-time PCR, (Excel Scientific cat#TS-RT2-100)
• Training on the ABI qPCR machines in MBB (contact the Core for information).

Protocol

1. Primer Design
2. RNA Preparation
3. Reverse Transcription
4. qPCR

1. Primer Design

Design primers to your target(s) and four reference genes. You will eventually use at least 2 of these reference genes.

Reference genes:

• You will need to find multiple reference genes to normalize expression to.
• These need to be stable across all the conditions (control and experimental) and this needs to be empirically determined by your own testing (see later).
• Candidate genes might come from:
  • Previous publications
  • Previous experience
  • Microarray compendiums; variance in expression can be determined for individual genes from thousands of samples across multiple conditions (e.g. Faith et al, 2008 Nucleic Acids Research, 10.1093/nar/gkm815).

Design process:

• Go to http://www.idtdna.com/primerquest/home/index, enter your sequence and select ‘two primers, intercalating dye’.
• Click on ‘customize your assay’. Up the optimal Tm to 63 and the minimum Tm to 60.
• Increase the optimal amplicon length to 120bp.
• Click ‘get assays’ and find a primer pair that closely matches the above conditions and for which both the primer Tms are equal. Click ‘view assay details’. Hovering over the primer sequence will give you options to check secondary structure and BLAST for off target transcripts. Do this.
• Order your favorite primer pair.

2. RNA Preparation

Prepare RNA as detailed here or here, ensuring that you include on column DNase digestion. NOTE: column digestion of DNA is not always 100% complete. If you find significant DNA contamination in your RNA samples (see later), you need to complete DNase treatment using DNase 1 and use a column to clean up the RNA from the reaction mix.

3. Reverse Transcription

Use the high capacity reverse transcription kit from ABI.

Per sample:

Conditions:

• 25°C – 10 min
• 37°C – 120 min
• 85°C – 5 min

Store cDNA at -20°C, or for longer durations, -80°C

4a. Choose your own qPCR Adventure

Choices before starting

In order to perform the qPCR that will answer your biological questions, you will first need to go through a few rounds of qPCR optimization. The amount of preliminary qPCR plates you will need to set up before you do your experimental qPCR will vary based on what you've done before. Refer to the flow chart on the right to figure out the path that you should take and then follow the corresponding link below to learn about the details of each step!

• Primer Efficiency qPCR -- Determine the efficiency of the primers you designed
• cDNA Concentration qPCR -- Determine the optimal concentration of cDNA to use in your qPCRs
• Reference Gene qPCR -- Determine the most consistent ref gene for your conditions
• Experimental qPCR -- Run your experiment

4b. qPCR Reaction Setup

NOTES

• The degree of dilution for the cDNA will be determined in one of your initial qPCR runs.

5. Load 3ul of sybrgreen/primer mix to each well. You do not need to change tips between wells for the same mix, only between mixes. It is important to accurately load each well:

• Lower the tip to the bottom of the well
• Lightly touch the bottom
• Eject the total contents
• Keep the plunger depressed as you pull out of the well.
• Do not drag the tip up the sides of the well when pulling out.