I. Preparation of total RNA
Reagent - TRIzol Reagent (Cat# 15596-018) - Invitrogen
Have used for preparation of total RNA from a variety of mouse (adult brain, liver, spleen, kidney, testis, ovary, heart and lung; embryonic brain, liver, kidney, spleen; mouse embryonic fibroblasts) and human (cell culture, T cells) tissues
II. Reverse transcription reaction
Kit - Superscript First Strand Synthesis for RT-PCR (Cat# 11904-018)- Invitrogen
Reverse transcribe 5 ug total RNA
primed with 200 ng random hexamers
Reaction mixture - 1X RT Buffer[20mM Tris-HCl, pH8.4, 50mM KCl], 2.5mM MgCl2, 10mM DTT, 500 uM each dNTP
1. Add 5 ug total RNA + 200 ng random hexamers, incubate (10min, 70C), chill on ice for 1 min
Kit - SYBR Green PCR Core Reagent (Cat# 4304886) - Applied Biosystems
Accessory Reagents -
iCycler iQ 96 well PCR plates (Cat# 2239441) (for BioRad system)
iCycler iQ Optical tape (Cat# 2239444) (for BioRad system)
Fluorescein Calibration Dye (1mM in DMSO, Cat# 170-8780) (for BioRad system)
MicroAmp Optical 96 well Rxn Plate
(Cat#N801-0560) - Applied Biosystems (for ABI system)
MicroAmp Optical Caps (Cat#N801-0935) - Applied Biosystems (for ABI system)
We use the SYBR Green assay. SYBR Green is a dye which binds indiscriminately to double-stranded DNA. (The actual Taqman assay involves the use of a dye-labeled probe which binds to the intervening sequence between the two primers; its removal during amplification is responsible for a change in fluorescence.) The advantage of the SYBR Green assay is that it does not require the use of the dye-labeled probe. It does, however, require the amplicon to be a single, clean product. The change in fluorescence required for real-time analysis is the result of SYBR Green binding to increasing amplified product.
1X SYBR PCR Buffer
200uM each dNTP
Units AMP Erase UNG (for ABI system only) for Bio-Rad iCycler add 10nM fluorescein for internal standard.
100 nM forward primer
100 nM reverse primer
1.25 Units AmpliTaq Gold polymerase
25 uL reaction volume (24 uL rxn mixture + 1 uL cDNA)
Primer design (characteristics):
50% G or C residues
amplicon size between 100 - 150 bp
cross intron-exon boundary
40 cycles of 95C-15sec, 60C-1 min
1. Establish working range within which your gene of interest falls.
We began by diluting our RT product for 5 points of analysis (1:10, 1:30, 1:100, 1:300, 1:1000) to allow us to generate a standard curve. Assuming a 100% efficient RT reaction, the range over which this curve lies is from approximately 8 pg > 8 ng of cDNA. We dilute our unknown samples (for most genes) 1:10 or 1:100 which allows us to quantify using the above standard curve.
We try to use the same threshold value (0.100 for ABI or 100 for BioRad) for each analysis which allows us to maintain consistency in analysis from run to run.
2. Establish comparably efficient reactions between control gene and gene of interest.
We use beta-2-microglobulin as our control gene for both mouse and human PCR. Since the use of SYBR Green requires two separate reactions (gene of interest and control), we normalize our results to beta-2-microglobulin to to minimize sample-to-sample variability. Other possible ubiquitously expressed control genes may include GAPDH, cyclophilin, 28S rRNA, etc.
It is important that the efficiency (determined by the slope of the curve generated) for your gene of interest and the control gene are similar. This will allow you to use the delta Ct method of analysis in a particular run which is merely a subtraction of the Ct values for the 2 genes. This saves you from having to run a standard curve in each reaction.
3. Analysis via a relative standard curve.
Use serial dilutions of the RT rxn, as described above, (ex. 1:30, 1:100, 1:300, 1:1000 etc.) to derive a relative value which can be used to compare WT vs. mutant or treatment type, or whatever you may want to directly compare. Calculate the relative values of your unknowns vs. the relative standard curve.
4. Analysis via an absolute standard curve.
Due to the lack of long-term
stability (repeated freezing-thawing, etc.) of a particular RT product, the
best, most reproducible standard curve can be derived from a (plasmid) DNA
curve. Prepare plasmids containing
your amplified segment Use a
standard curve of known amounts of a specific plasmid (ex. 25 pg, 0.25 pg,
0.025pg, etc.) for both the control gene and the gene of interest. An absolute standard curve would allow
you to quantify your expression on the basis of cell number or other method.
Primers used in Gronostajski lab:
mb2mg B2MM AGACTGATACATACGCCTGCAG 119bp product
mA total mNFI-AE23 TGGCATACTTTGTACATGCAGC 128bp product
mB total mNFI-BE2 GTTTTTGGCATACTACGTGCAGG 133bp product
mC total mNFI-CE22 GACCTGTACCTGGCCTACTTTG 147bp product
mX total mNFI-XE2 CTGGCTTACTTTGTCCACACTC 148bp product