Recommended RNA Purification Kit

October 05, 2026

BlueskyReddit
Stanford University
Biochemistry
Senior Research Scientist

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Company:

Qiagen

Product Name:

RNAeasy Mini Kit

Catalog Number:

74104

WNT signaling is essential for embryonic development and adult tissue homeostasis, and its aberrant activation drives several cancers, most notably colorectal cancer. My lab studies the molecular mechanisms of oncogenic WNT signal transduction, with the goal of identifying therapeutic vulnerabilities in colorectal cancer. We edit endogenous pathway genes using Cas9 RNP electroporation and synthesize sgRNAs in-house by in vitro transcription. I use the Qiagen RNeasy Mini Kit (Cat# 74104) to purify those sgRNAs, following a protocol modified for small RNAs.

Experimental Design and Results Summary

Application

Molecular Biology

Starting Material

sgRNA generated by in vitro transcription

Tips

None

Results Summary

I use RNeasy mini spin columns to purify ~100 nucleotide sgRNAs from overnight T7 in vitro transcription reactions after DNase and alkaline phosphatase treatment. The protocol is modified for small RNAs by increasing the ethanol concentration at the binding step, since the standard RNeasy procedure deliberately excludes RNAs below about 200 nucleotides. Reactions are bound in RLT buffer with added ethanol, washed twice with RPE, and eluted in 30–50 µL of DEPC-treated water. In the experiment shown here, two sgRNAs were purified in parallel and recovered at ~2000 ng/µL. To confirm that the purified guides were intact and functional, I assembled each with recombinant Cas9 and tested cleavage of a PCR amplicon spanning the target site. Both sgRNAs cleaved the substrate efficiently, yielding the expected product sizes, while the no-guide control was uncut. The same preparations were then used directly for Cas9 RNP electroporation into HAP1 cells, and I use this workflow routinely for in-house guide production.

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Summary

The Good

The main strength is that a standard, widely stocked RNA cleanup kit can be adapted to recover short sgRNAs with a single change to the binding conditions, avoiding the cost of a dedicated small-RNA kit. Yields are more than sufficient. Elution into 30–50 µL of water produces stocks concentrated enough to use directly in RNP reactions without a further concentration step, and the eluate was clean enough for electroporation into cells. The columns accept the DNase- and phosphatase-treated transcription reaction directly.

The Bad

None

The Bottom Line

With a simple modification of the binding step, the Qiagen RNeasy Mini Kit is a practical and economical way to purify in vitro transcribed sgRNAs for CRISPR work. Yields are ample and the recovered RNA performs well in Cas9 RNP electroporation. For labs producing their own guides, I recommend it.

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