TITLE: Rat Brain Tissue RNA Extraction/cDNA Synthesis for qPCR
AUTHORS: Kokila Shankar, Olivier George

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is to obtain cDNA from rat brain tissue micropunches for downstream qPCR. Briefly, RNA will be extracted from brain tissue and will be converted to cDNA through reverse transcription reaction. Protocols have been adapted from Zymo (</span><a href="https://files.zymoresearch.com/protocols/_r2060_r2061_r2062_r2063_direct-zol_rna_microprep.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://files.zymoresearch.com/protocols/_r2060_r2061_r2062_r2063_direct-zol_rna_microprep.pdf</span></a><span>) and New England Biolabs (</span><a href="https://www.neb.com/protocols/2013/01/23/first-strand-cdna-synthesis-protocols-e6560" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.neb.com/protocols/2013/01/23/first-strand-cdna-synthesis-protocols-e6560</span></a><span>). </span></div></div>

[STEPS]
?. Remove tissue samples from   and thaw on ice
-80 °C
?. If samples are stored in RNALater ICE, remove from tube and add  to each sample. Pipet homogenize until tissue is fully dissolved and leave at RT while working on other samples. Spin samples at 13000 RPM at  for   and transfer supernatant to new tube.
300 µl
4 °C
If samples were stored directly in Trizol, ensure tissue is fully dissolved in Trizol, and spin down as directed.
?. Add to each sample, mix well (invert tube, do not pipet), and transfer mixture to spin columns (with collection tube underneath). Spin samples at 13000 RPM at room temperature ( ) for   and transfer column to new collection tube.
300 µl
21 °C
?. Add  Direct-zol RNA Prewash buffer to each column. Spin samples at 13000 RPM at room temp ( ) for   and discard flowthrough. Repeat this step (2 total washes).
400 µl
21 °C
?. Add  RNA Wash buffer to each column. Spin at 13000 RPM at room temp () for  . Spin one more time for  to ensure all liquid is removed from column.
700 µl
21 °C
?. Add directly to column matrix (be careful not to pierce column) and spin at 13000 RPM at room temp ( ) for  . IMMEDIATELY TRANSFER COLUMNS TO ICE. Quanitify RNA concentration and purity (260/280) using
8 µl
21 °C
Make sure all RNA samples stay on ice and as sterile as possible to avoid RNase contamination. Move immediately to next step to limit RNA degradation.
?. On ice, in separate 200 uL PCR strip tubes, mix up to  RNA (up to  ) with  d(T)23VN and (if needed) to make an  total reaction. Mix samples well and make sure nothing is sticking to the sides of the tube. Make 1 additional tube to serve as negative control.
1 µg
6 µl
2 µl
8 µl
Master mix of d(T)23VN and/or H2O should be made to avoid pipetting in and out of stock tube
?. Denature RNA at  for  and cool down at  for  . Use preset "cDNAincubate" function on . Immediately put on ice.
70 °C
4 °C
?. To each sample tube, add  M-MuLV Reaction Mix and  M-MuLV Enzyme Mix. To the negative control tube, add  M-MuLV Reaction Mix and . Total reaction volume for all samples should be  .
10 µl
2 µl
10 µl
2 µl
20 µl
Ensure this step is done on ice
?. Incubate samples at  for  and then inactivate enzyme at  for  . Use preset "NEB cDNA" program on
42 °C
80 °C
At this point, samples can either be used directly in qPCR assay or stored for later use at