- Transcriptome Analysis: Library Construction 15
- Remove the sample tube from the magnetic stand and add 50μLofELB.
Gently pipette the entire volume up and down 10 times to mix thoroughly. - Place the sample on the thermal cycler to elute mRNA from the beads
using the following program:
80 ◦Cfor 2 minutes, 25 ◦Chold - Remove the sample tube from the thermal cycler and add 50μL of BBB.
Gently pipette the entire volume up and down 10 times to mix thoroughly.
Note: This allows mRNA to specifically rebind the beads, while reducing the amount
of rRNA that non-specifically binds. - Incubate the sample at room temperature for 5 minutes.
- Centrifuge briefly and place the sample on magnetic stand at room
temperature for 5 minutes. - Remove and discard all the supernatant from each sample.
- Remove the sample tube from the magnetic stand and wash the beads
with 200μL of BWB. Gently pipette the entire volume up and down 10
times to mix thoroughly. - Centrifuge briefly and place the sample on magnetic stand at room
temperature for 5 minutes. - Remove and discard all of the supernatant from each sample.
Note: The supernatant contains residual rRNA and other contaminants that were
released in the first elution and did not rebind the beads.- Remove the sample tube from the magnetic stand and add 19.5μL of EPF
to each sample. Gently pipette the entire volume up and down 10 times
to mix thoroughly.
Note: The EPF contains random hexamers for RT priming and serves as the 1ststrand
cDNA synthesis reaction buffer. - Place the sample on the thermal cycler to elute, fragment, and prime the
RNA from the beads using the following program:
94 ◦Cfor8minutes, 4 ◦C hold - Remove the sample from the thermal cycler, centrifuge briefly, and proceed
immediately to 1ststrand cDNA synthesis.
III.RT-PCR: 1ststrand synthesis
- Centrifuge briefly and place the sample on magnetic stand at room
temperature for 5 minutes. - Transfer 17μL of the supernatant (fragmented and primed mRNA) from
each sample to the corresponding new PCR tube.