RNeasy Mini Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- For purifying RNA add either 10 μl β-mercaptoethanol (β-ME) or 20 μl 2 M dithiothreitol (DTT). - To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).	- Include DNAse treatment for 15-20min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C. - Use water to elute the RNA that is warmed to ~60`C.

Protocol tips
- For purifying RNA add either 10 μl β-mercaptoethanol (β-ME) or 20 μl 2 M dithiothreitol (DTT). - To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).
Downstream tips
- Include DNAse treatment for 15-20min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C. - Use water to elute the RNA that is warmed to ~60`C.

Publication protocol

RNA was extracted and purified using the RNeasy mini kit. 0.5 μg of RNA was reverse transcribed using qScript cDNA synthesis kit (Quanta Biosciences). Connexin primers were designed using Primer-Blast yielding the following sense and anti-sense sequences: p53, forward-GTTCCGAGAGCTGAATGAGG, reverse-TTATGGCGGGAGGTAGACTG; p21, forward-GGAAGACCATGTGGACCTGT, reverse-GGCGTTTGGAGTGGTAGAAA. Values were normalized by β-actin, forward-TCACCCACACTGTGCCCATCTACGA, reverse-CAGCGGAACCGCTCATTGCCAATGG. All primers were tested using standard curves with 10-fold serial dilutions. The qPCR was performed in the CFX96 Real-Time PCR Detection System (Bio-Rad) using Perfecta Sybr Green Fastmix (Quanta Biosciences), and the data analyzed with CFX Manager software (Bio-Rad).

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Manufacturer protocol

Download the product protocol from Qiagen for RNeasy Mini Kit below.

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