RNeasy Mini Kit

RNA isolation / purification Cells - immortalized BHK-21

Experiment
RNA isolation / purification Cells - immortalized BHK-21
Product
RNeasy Mini Kit from Qiagen
Manufacturer
Qiagen

Protocol tips

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.

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Discussion

Discussion

2 years ago

Author: Switzerland

I am having trouble using the QiageN RNAeasy mini kit for CD34+ cells. Any tips?

I am having trouble using the QiageN RNAeasy mini kit for CD34+ cells. Any tips?

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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.

Paper title
Developing a Triple Transgenic Cell Line for High-Efficiency Porcine Reproductive and Respiratory Syndrome Virus Infection
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Manufacturer protocol

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

Download PDF Download manufacturer protocol

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