RNA isolation and cDNA synthesis from B-cells 三种B细胞

Pellet cells by centrifugation and add 1 ml TRIzol (1ml/1E7 cells). Pipet up and down to lyse cells. Use 800 ?l for less than 1E7 cells.

Incubate samples for 5 min. at room temperature to allow dissociation of nucleoprotein complexes. Add 200 ?l chloroform (per 1 ml TRIzol). (Use 160 ?l for less than 1E7 cells). Shake vigorously by hand for 15 seconds and incubate at room temperature for 2 ? 3 min.

Centrifuge no more than 11,500 rpm for 15 min. at 4C. The RNA will be found exclusively in the aqueous phase which will be about 60% of the volume of TRIzol used.

Transfer to fresh tube and precipitate with 1 ?l glycogen and 500 ?l isopropanol (per 1 ml TRIzol). Incubate at room temperature for 10 min and centrifuge at 12k g for 10 min at 4C.

Decant supernatant and wash with 1 ml 75% EtOH. Vortex and centrifuge at 9,000 rpm for 5 min at 4C.

Decant and air-dry pellet.

Dissolve in RNase-free H2O (40 ?l for 1E7 cells or more and 20 ?l for less)

If there is less than 5 ?g of RNA generated, then use the speed-vacuum to remove most of the H2O to accommodate the cDNA reaction.

Use 5 ?g total RNA/reaction, 1 ?l oligo dT primer (100 pmol/?l), 10 ?l RNase-free H2O. Total volume = 11 ?l

Heat reaction at 70C for 10 minutes and quick chill on ice. Centrifuge briefly and add: 4 ?l 5x First-strand reaction buffer, 2 ?l 0.1 M DTT, 1 ?l 10 mM dNTP mix. Vortex and centrifuge briefly. Place tube at 45C for 2 min to equilibrate before adding reverse trancriptase.

Add 1 ?l SuperScript II RT (total vol. ~ 20 ?l) and incubate aqt 45C for 1 hours.

Place tube on ice to terminate reaction or store in freezer (-20C).

Use 1 ?l per reaction.