RNeasy Mini Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- 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. - For purifying RNA add either 10 μl β-mercaptoethanol (β-ME) or 20 μl 2 M dithiothreitol (DTT).

Protocol tips
- 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. - For purifying RNA add either 10 μl β-mercaptoethanol (β-ME) or 20 μl 2 M dithiothreitol (DTT).

Publication protocol

Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Cat. 74104) following the manufacturer’s instructions. Genomic DNA was removed using DNase I (Qiagen, Cat. 79254). A total of 2 μg of RNA was reverse transcribed for first-strand cDNA synthesis using Moloney murine leukemia virus transcriptase (Promega, Cat. 28025–013) and the oligo (dT) 18 primer. Quantitative RT-PCR (qPCR) analysis was performed using the Roche LightCycler 480 System (LC 480; Roche, Basel, Switzerland) and different primers. ORF-7 was used to determine the level of PRRSV mRNA expression. GAPDH from BHK-21 cells (hGAPDH) or MARC-145 cells (sGAPDH) was used as an internal reference gene. To detect IFN-β and IFN-stimulated gene (ISG) expression in infected BHK-21, BHK-21-TTG, and MARC-145 cells, the primers were synthesized and listed in Table 2. Absolute qPCR was used to detect the copy number of PRRSV in the supernatant. Meanwhile, a standard set of mixtures (representing 1, 2, 4, 8, 16, and 32 copies of plasmid DNA consisting of ORF7 sequence) was used to generate a standard curve to determine the correlation between the Ct value and the copy number of virions.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing RNA isolation / purification Cells - immortalized BHK-21 using RNeasy Mini Kit from Qiagen.

Manufacturer protocol

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

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing RNA isolation / purification Cells - immortalized BHK-21 using RNeasy Mini Kit from Qiagen. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.