Microarrays and Q-PCR | Home |
RNA preparation
Trizol and Qiagen column procedures have yielded good results.
Tri Reagent from MRC, a reagent similar to Trizol, is thought to yield quality better RNA, and certainly does yield much less genomic DNA (important for Q-PCR). In addition, the extraction with this improved reagent is with bromochloropropane, which is less toxic than chloroform.
TURBO DNA- free kit from Ambion works very well for removing Genomic DNA from RNA, which is particularly important for Q-PCR. Heat-killing of DNase in Ca++ and Mg++ containing buffer prior to cDNA synthesis can severely degrade the RNA. This kit quickly removes the modified DNase by binding it to a resin—no fuss, no muss.
Storage of tissue or cells prior to RNA isolation
RNAlater from Ambion is a reagent available from Ambion which allows harvested tissues or cells to be safely stored prior to RNA extraction, without degradation of the RNA.
RNAlater-ICE is a similar RNA protecting reagent that can be added to deep frozen samples prior to thawing and processing. Both types of samples can then be processed by Trizol or Tri Reagent extraction.
RNA quality
Desired quality analysis of RNA prior to microarrays is a determination of the A260/A280 ratio (should be close to 2.0 – the Nanodrop spec is handy for such analysis), and standard TBE agarose minigel analysis. The latter, using 1 µg of RNA and an SDS washed gel box, should yield clear 28S and 18S ethidium stained rRNAs, with the 28S band brighter than the 18S band. An Agilent bioanalyzer or Biorad Experion is more elegant for QC of RNA, but we do not currently have such instruments.
Microarrays
An Ambion RNA amplification kit is used for making biotinylated cRNA, based on the method originally described by Eberwine. For patent reasons, the instruction manual of the labeling kit (pdf) does not describe the final step for making labeled cRNA, which is incubation of the biotinylated cRNA with streptavidin-Cy3
Illumina on-line tutorial for microarrays, describing the technology, hardware, and software.
Illumina PDF bulletin on whole genome expression profiling
Q-PCR
Comprehensive Stratagene guide to real-time PCR (large PDF file)
Brochure for our MX3000p (real-time PCR instrument)
Primer 3 free web-based software for primer design. Used for all Facility-designed primers, with following changes to the defaults:
Change Tms to: low: 54, Opt. 56, High 59
Change: max 3' comp. =2, max polyX=4. GC clamp=1, (all in right hand column)
Set repeat seq screen to correct organism (top right).
Full information on finding cDNA sequences and primer design
Microtiter format excel template for setting up experiments
Other useful links
Local array core facilities (we welcome comparison shopping!)
Real-time PCR
Roche Lightcycler and reagents
RNA and cDNA suppliers
Ambion (variety of normal and diseased RNA, even tumor/adjacent normal)
Clontech (now part of BD) many RNAs, cDNAs, also universal ref. RNA and universal ref. cDNA from tissues (as opposed to multiple cell lines like Stratagene)
Stratagene (wide assortment), including Universal Reference RNA (Human, mouse, or rat—pooled RNAs representing nearly all genes --very useful for Q-PCR and microarrays)
SuperArray (reference RNA from 20 tissues - human, mouse, or rat)
Databases
Source Site The best starting point for information on a gene – batch query too
TIGR human and TIGR mouse databases – shows consensus of all ESTs
ENSEMBL Human and ENSEMBL Mouse browsers – vast genomic resources
RIKEN Full length cDNA resources, particularly mouse
Entrez Now the central search engine across NCBI
GeneCards database of human genes and their involvement in disease
KEGG pathways The central database for genetic and metabolic pathways
Microarray Data