Absolute Quantification
|
Absolute Quantification |
((Prepare this in your own lab):
Designing an Absolute Quantification Experiment
Overview:
Typical AQ exoerunebts are designed for traditional (singleplex) PCR, where a single primer pair plus a TaqMan proble or SYBR binding dye are present in a reaction. The following sections describe decisions required for AQ experiments.
Specifying the Components of an AQ Experiment
for each AQ experiment, specify:
An unkown - The nucleic acid sequence that you are quantifying.
Standards - This guide assumes that you have generated a set of standards for each target sequence that you are quantifying.
Replicate wells - For absolute quantification assays, use three or more replicate reactions per sample to ensure statistical significance.
Selecting the Chemistry:
1. TaqMan Reagents or Kits
or
2. SYBR Green Reagents
Selecting One- or Two-Step RT-PCR
One-Step: Perform RT and PCR in a single reaction.
Two-Step: Perform RT and PCR in separate reactions.
CONVERTING RNA TO C-DNA
1. Isolating Total RNA
2. Quality of RNA: have an A260/280 greater than 1.9, be intact wehn visulaized by gel electrophresis, not contain RT or PCR inhibitors.
3. Starting Concentration of total RNA should be 0.1-10 ug.
4. Reverse Transcription (1x):
10 ul 10 x RT Buffer
4 ul 25x dNTPs
10 ul 10x random primers
5 ul MultiScribe Reverse Transcriptase
21 ul Nuclease-free water
Total: 50 ul
25 C for 10 min, then 37 C for 120 min. Store all cDNA samples at -15 to -25 C.
RUNNING AN AQ PLATE
1. Preparing the PCR Master Mix
2. Preparing the Reaction Plate
Take your plate to 33 Life Science Building, Auburn University Genomics and Sequencing Laboratory: