Griffin:Nested RT-PCR

Nested PCR overview
Santa Cruz Biotechnology inc. offers nested primers for measuring transcript levels. Each product will have 2 vials, an A set (forward & reverse) and B set (forward & reverse) primers. Each set of primers is provided 20 µl at a concentration of 10 µM. Since the primers are designed toward an mRNA template, they are designed to cross exon junctions and will not amplify genomic DNA.

The amplification of RNA requires the conversion of the RNA substrate into DNA. This is achieved through the use of a reverse transcriptase such as AMV RT (avian myeloblastis virus reverse transcriptase) or M-MuLV RT (moloney murine leukemia virus reverse transcriptase). The resulting cDNA can be used as a template for a standard PCR.

Nested PCR means that two pairs of PCR primers were used for a single locus. The first pair (A set) generates an amplicon within the locus as seen in any PCR experiment. The second pair of primers (B set; nested primers) bind within the first amplicon and produce a second PCR product that will be shorter than the first one. The logic behind this strategy is that if the wrong locus were amplified by mistake, the probability is very low that it would also be amplified a second time by a second pair of primers.

A set primer amplicon size ~1000 bp

B set primer amplicon size 250-500 bp


 * To identify low levels of DNA contamination, do a PCR of a housekeeping gene and a portion of the RNA preparation as template. If there is contamination, there will be products in all samples.

Primer Tm Values
Tm values for PCR primers range between 55-60 C (19-22 nt, GC% ~55%, no Salt) OR 63-68 C w/salt. The A and B nested primer sets share similar base pair length, GC% and Tm values.

Nested PCR utilizes two pairs of PCR primers for a single locus. The first primer pair A set amplifies within the locus. The second primer pair B set (nested primers) then binds within the 'A' amplicon to produce a second nested 'B' amplicon.

Reagents

 * Reverse Transcriptase
 * Deoxynucleotide Mix, dATP, dCPT, dGTP, dTTP, 10 mM each, in sterile double-distilled water, pH 8.5
 * Reaction Buffer, 10x conc., 1.05 ml; 100 mM Tris-HCl, 500 mM KCl, pH 8.3 (20°C)
 * MgCl2 Stock Solution, 2 x 1.3 ml each 25 mM MgCl2
 * Gelatin, 0.05% gelatin (w/v)
 * Oligo-p(dT)15 Primer, 0.02 A260 units/µl (0.8 µg/µl)
 * RNase Inhibitor, 50 U/µl
 * Sterile Water

PCR Optimization

 * MgCl2 concentrations may vary depending on the template, primer, and dNTP concentrations in the amplification reaction. To optimize conditions, use a MgCl2 titration, generally between 0.5 and 10 mM.
 * Primer concentrations may vary; typical final concentrations range from 0.01 to 0.5
 * The amount of cDNA utilized in RT-PCR reactions may vary depending on the nature of the RNA template; typically, 5 ul of the cDNA of reverse transcribed total RNA, 20 ul of the cDNA resulting from reverse transcribed poly(A)+ RNA, or 20
 * Addition of Gelatin (0.01 mg/ml final) stabilizes Taq DNA polymerase during the PCR reaction, yielding more amplification product.

Internal Control
Primers toward an existing transcript or RNA entity will ensure when reaction conditions yield signal.


 * GAPDH
 * 18s rRNA

cDNA Synthesis (Reverse Transcription)
In a standard RT-PCR assay, varying amounts of RNA template 10ug, 1ug, 100ng, 100pg are reversely transcribed with a poly dT primer that attaches to the polyadenylation track on mRNA to yield a cDNA template.

a) 1 ul oligo (dT)12–18 (500 ug/ml)
 * Prepare a solution containing:

b) 1 ng-5 ug total RNA

c) 1 ul 10 mM dNTPs

d) and add RNase-free water to a final volume of 12 ul


 * If extensive secondary structure is potentially present in the RNA, the RNA sample may be denatured at +70°C for 5 min before adding it to the reaction minimize RNA secondary structure, and placed on ice for 5 min before adding it to the reaction.

a) 4 ul 5x reverse transcriptase buffer

b) 2 ul 0.1 M DTT

c) 1 u RNase inhibitor

Reverse Transcritpion Reaction

 * Incubate at +30°C for 10 min minutes to anneal primer and template.


 * Add 1 ul reverse transcriptase (200 units) and incubate at 42° C for 60 minutes to extend the primer and then terminate the reaction by incubating at 70° C for 15 minutes. The RNA is subsequently reverse transcribed, resulting in cDNA synthesis.

NOTE: (As an optional step add 1 ul RNase H (2 unit/ul) and incubate at 37° C for 20 minutes)

First PCR reaction

 * Prepare a solution containing:

a) 5 ul 10x PCR buffer (with or without MgCl2)

b) 5 ul 25 mM MgCl2 (It may be necessary to vary the MgCl2 concentration, 2.5 mM final concentration recommended.)

c) 1 ul 10 mM dNTP

d) 1 ul primer pair A

e) 1 ul Taq DNA polymerase

f) 2 ul cDNA and add water to 50 ul

First PCR amplification parameters
Perform 15–35 cycles of PCR. Annealing and extension conditions are primer and template dependent and must be determined empirically for each template-primer pair. Tm values for PCR primers generally range between 55-60 C


 * Denaturation 96°C, 0.5-1.5 minute
 * Annealing 57°C, 0.5-1 minute
 * Polymerization (35 cycles) 72°C, 0.5-2 minutes
 * Link; Extension (1 cycle) 70°C, 5 minutes
 * Link to a 4°C Soak file.

NOTE: Addition of Gelatin (0.01 mg/ml final) stabilizes Taq DNA polymerase, yielding more amplification product.

Second (nested) PCR reaction

 * Prepare a solution containing:

a) 5 ul 10x PCR buffer (with or without MgCl2)

b) 5 ul 25 mM MgCl2 (It may be necessary to vary the MgCl2 concentration, 2.5 mM final concentration recommended)

c) 1 ul 10 mM dNTP

d) 1 ul primer pair B

e) 1 ul Taq DNA polymerase

f) 1–5 ul first PCR product and add water to 50 ul

Second PCR amplification parameters
Perform 15-25 cycles of PCR. Annealing and extension conditions are primer and template dependent and must be determined empirically for each template-primer pair. Tm values for PCR primers generally range between 55-60 C


 * Denaturation 96°C, 30 sec
 * Annealing 57°C, 30 sec
 * Polymerization (35 cycles) 72°C, 30 sec
 * Link; Extension (1 cycle) 70°C, 5 minutes
 * Link to a 4°C Soak file.

NOTE: Addition of Gelatin (0.01 mg/ml final) stabilizes Taq DNA polymerase, yielding more amplification product.


 * PCR products are visualized by agarose gel electrophoresis stained with an appropriate dye.

Agarose Gel Electrophoresis
Agarose gel electrophoresis can resolve DNA or RNA by size. DNA/RNA is visible in the gel when ethidium bromide is added during the gel casting process. EtBr intercalates between dsDNA or dsRNA and absorbs invisible UV light and emits visible orange light. SYBR Green absorbs blue light (λmax = 488 nm) and emits green light (λmax = 522 nm) may be used in place of EtBr, although more commonly this dye is used for qPCR.


 * 0.7% gel will show good separation (resolution) of large DNA fragments (5–10kb)
 * 2% gel will show good resolution for small fragments (0.2–1kb)
 * 3% gel or vertical polyacrylamide gel for separating smaller fragments (the higher the % gel, the more brittle the gel)

Gel Casting
The volume of agarose required for a minigel is around 30–50mL.


 * Weigh out 0.5g of agarose into a 250mL conical flask. Add 50mL of 0.5xTBE, swirl to mix.
 * Microwave for about 1 minute to dissolve the agarose. Use gloves to handle and do not let it overboil; molten agarose is very hot.
 * Let cool for 5 minutes or warm to touch with bare hands.
 * Add 1µL of ethidium bromide (10mg/mL) and swirl to mix.
 * Pour the gel into the resevoir. Push any bubbles away to the side using a disposable tip. Insert the comb. Wait at least 30-60 min.
 * Add 0.5x TBE buffer into the gel tank to submerge the gel to 2–5mm depth. This is the running buffer.
 * Load samples and run the gel no greater than 5 Volt/cm.

2L of 10xTBE

 * 218g Tris base
 * 110g Boric acid
 * 9.3g EDTA

Dissolve the ingredients in 1.9L of distilled water. pH to about 8.3 using NaOH and make up to 2L.

Loading buffer

 * 25mg bromophenol blue or xylene cyanol (Bromophenol blue migrates @ ~200–400bp. Xylene cyanol migrates ~4kb)
 * 4g sucrose
 * H2O to 10mL

Store at 4°C or -20