[MagNA Pure LC RNA Isolation Kit – High Performance (Roche Applied Science), alternatively other RNA isolation protocols may be utilized]
(a) Ensure that the MagNA Pure LC Instrument has the proper RNA purification protocols installed as detailed by the MagNA Pure LC RNA Isolation Kit manual.
(b) Prepare the isolation kit reagents as detailed in the manual.
(c) Aliquot 200 µl of whole blood or white blood cells, or 1 x 106 peripheral blood mononuclear or cultured cells as directed by the manual.
(d) Set up the instrument using the appropriate protocol for the starting material. Follow instructions supplied by the software, as it will calculate reagent volumes based upon number of samples to be isolated.
(e) Immediately upon completion of the isolation run, quantify RNA samples with a NanoDropTM (Thermo Scientific) prior to proceeding with cDNA synthesis. If necessary, RNA samples can be stored in RNase-free microcentrifuge tubes at -80 °C.
[MJ Research Tetrad Thermocycler (Bio-Rad Laboratories) and Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen), alternatively other thermocyclers may be utilized.]
(a) Prepare a reaction mixture of 1 µl 10 mM dNTP mix, 1 µl 50 µM Oligo(dT)20, and 50 ng total RNA (or up to 8 µl if 50 ng RNA would exceed 8 µl). If necessary, add DEPC-treated water to obtain a 10 µl final reaction volume.
(b) Incubate reactions at 65 °C for 5 min to denature. During this incubation, prepare the cDNA synthesis master mix of 2 µl 10X RT Buffer, 4 µl 25 mM MgCl2, 2µl 0.1 M DTT, 1 µl RNaseOUTTM, and 1 µl SuperScriptTM III RT.
(c) Incubate initial reaction mixture at 4 °C or on ice for at least 1 min. During this incubation, add 10 µl cDNA synthesis master mix to initial reaction mixture.
(d) Incubate at 50 °C for 50 min.
(e) Terminate cDNA synthesis by incubating at 85 °C for 5 min.
(f) Add 1 µl 2U/µl E. coli RNase H to reaction mixture and incubate at 37 °C for 20 min to remove any remaining RNA.
(g) Upon completion, proceed directly to PCR or store cDNA at -20 °C.
[Phusion Hot Start DNA polymerase (New England BioLabs)]
(a) Prepare 20 µl primer-specific reaction mixtures containing 1 µl cDNA, 0.5 µM F/R PCR-SSP primers, 0.4 U Phusion Hot Start DNA polymerase, high fidelity buffer, 2% DMSO (NEB) and 200 µM each dNTP (Roche).
(b) Use the the following cycle conditions: 98°C for 60 s; 25 cycles of 98C for 5 s, 67-69°C* for 1 s, 72°C for 20 s; 72°C for 5 minutes.
*67°C for B*440101, B*460101, B*510101; 69°C for B*430101
[1x TAE, genetic analysis grade agarose, 6X loading dye, SYBR Safe DNA gel stain 10,000X concentrate in DMSO (Invitrogen), ø X174/HaeIII size ladder (Promega)]
(a) Prepare a 1.7% (w/v) agarose gel by combining 150 µl 1X TAE with 2.55 g agarose.
(b) Heat the mixture in the microwave to melt the agarose, swirling occasionally.
(c) Add 15 µl SYBR Safe DNA gel stain to the mixture, swirling to mix. Pour gel and allow to solidify.
(d) Add 4 µl 6X loading dye to each PCR reaction and load entire volume into a single well of the agarose gel. Space samples appropriately leaving at least one empty well between samples to avoid cross-contamination. Load 6 µl ladder into an empty well for sizing of the amplicon.
(e) Run agarose gel in 1X TAE buffer at 120 V for 1 h. Ensure that the products have sufficiently separated. Extend running time if necessary.
(f) Once sufficient separation is observed, visualize with AlphaImager program (UV transillumination)
Reference:
Mee ET, et al. MHC haplotype frequencies in a UK breeding colony of Mauritian cynomolgus macaques mirror those found in a distinct population from the same geographic origin. J Med Primatol. 2009 Feb;38(1):1-14. Epub 2008 Nov 5.
Karl JA, et al. Identification of MHC class I sequences in Chinese-origin rhesus macaques. Immunogenetics. 2008 Jan;60(1):37-46. Epub 2007 Dec 21.
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