Various ways to optimize RNA isolation

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RNA extraction is one of the most skilful jobs in the molecular biology world. It is critical to have a pure RNA sample as it determines the further outcome of molecular techniques such as reverse transcription real-time PCR (RT-qPCR), transcriptome analysis, array analysis, digital PCR, northern blotting, and cDNA library construction. Not yet there? Don´t worry yet! GENAXXON is the friend, you will know in these times. Although almost the entire process of the isolation needs precision and concentration, there are some steps that are of particular importance and optimising them can lead to a maximised yield and improved RNA quality. To know more check out our Troubleshooting guide. Find all our exciting RNA purification kits and products here! Do try our exclusive Viral RNA Purification Kit.

RNA isolation starts with tissue homogenization and cell disruption. There are various ways of doing this broadly divided into mechanical methods and enzymatic methods. The mechanical methods include homogenization with a mechanical homogenizer, vortexing, sonication, French press and bead milling. The enzymatic methods include Lysozyme, zymolase and lysostaphin digestion. Several things need to be kept in mind for example:-

1) The time and method of sample disruption
depends on sample size as an animal tissue sample requires more homogenisation time than drosophila sample.

2) The disruption should always be carried out on ice and a cold temperature
must be maintained throughout as RNA is heat labile.

3) The sample for RNA isolation must be disrupted in a way to maintain the integrity of RNA in it.
This should be taken particularly in consideration when enzymatic methods of disruptions are used.

Problem	Cause	Solution
Clogged column	Improper homogenization of the sample	Centrifuge well after the treatment with Proteinase K step. Do not include the interphase while picking up the sample for RNA extraction. Homogenize the sample properly by mixing with reagents and prolonging the homogenization time. Too many cell debris lead to clogging. Centrifugation should be carried out at RT as lower temperature may also result in clogging.
Clogged column	Excess sample	Sample amount should be added first in very low quantity in the beginning, so as to make the column wet and then step by step low volumes of sample should be applied on to it.
Low/no RNA yield	Improper elution	Prolong the incubation time of elution buffer in the column (atleast 5- 10 minutes) before centrifugation step. Add adequate amount of elution buffer or else thee buffer gets absorbed by the column only, resulting in lower yields.
	Improper homogenization of the sample	For successful RNA extraction, cells need to be disrupted, which is ensured by proper homogenization step.
	RNases were introduced during/after sample processing.	RNAse is present on our hands, hair, and other parts of the body as well as the work bench. Therefore it is necessary to clean the work surface before RNA extraction as well as to wear gloves at all times.
Flawed performance of extracted RNA in downstreaming processes	Presence of ethanol or salts	Make sure to get rid of all the ethanol and salts during washing, as they may interfere with the downstreaming process.

References:

1) Tesfamichael, D. H., Wood, M. W., & Pritchard, J. C. (2020). Comparison of commercial manual extraction kits for RNA isolation from canine whole blood. Journal of Veterinary Diagnostic Investigation. https://doi.org/10.1177/1040638720938026

2) Ourania Tsatas, Nader Ghasemlou. (2020). Isolation and RNA purification of macrophages/microglia from the adult mouse spinal cord.Journal of Immunological methods. https://doi.org/10.1016/j.jim.2019.112678

3) Cox A, Tolkach Y, Kristiansen G, Ritter M, Ellinger J. The lncRNA Fer1L4 is an adverse prognostic parameter in clear-cell renal-cell carcinoma. Clinical & Translational Oncology : Official Publication of the Federation of Spanish Oncology Societies and of the National Cancer Institute of Mexico. 2020 Sep;22(9):1524-1531. DOI: 10.1007/s12094-020-02291-0.