Assessment of Protein Profiles of RNAlater Stored and Fresh PBMC Cells Using Different Protein Extraction Buffers
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Assessment of Protein Profiles of RNAlater Stored and Fresh PBMC Cells Using Different Protein Extraction Buffers R. R. Alyethodi1 · S. Karthik1 · K. Muniswamy1 · S. K. Ravi1 · P. Perumal1 · D. Bhattacharya1 · P. A. Bala1 · A. K. De1 · T. Sujatha1 · Jai Sunder1 · A. Kundu1
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract For proteome analyses, the tissue samples are mostly preserved either snap frozen or formalin-fixed, paraffin-embedded form. Use of RNAlater—a non-toxic solution primarily used to stabilize the RNA content of samples—in tissue preservation for proteome analysis recently described equally reliable with snap-frozen preservation in human tissues. Even though RNALater storage has great potential in the preservation of Peripheral Blood Mononuclear Cells (PBMC), its impact on the results of proteome analysis is poorly described at qualitative and quantitative measures. The present study investigated protein profiles of RNAlater preserved and fresh PBMCs using three extraction buffers viz. Triton X-100, RIPA and SDS. Proteins are separated in SDS-PAGE and quantified using densitometry. On an average 19.3 bands from fresh and 15.6 bands from RNAlater storage cells were obtained with a molecular weight ranging from 25 to > 250 kDa. RNAlater storage generated a fewer number and lesser quantity of low molecular weight proteins while yielded a similar or high quantity of high molecular weight protein fractions. The principal component analysis showed that Triton X-100 is inferior as compared to SDS and RIPA with respect to their protein bands and quantity yielded. While RNAlater is effective in preserving PBMC for proteome analysis, our findings warrant caution in its use in proteomics experiments especially if the target is low molecular weight proteins. Keywords: RNAlater · PBMC · RIPA · SDS-PAGE · Densitometry · PCA
1 Introduction For the success of any proteomic experiments, sufficient quality and quantity of protein are essential. Snap-freezing in liquid nitrogen has been the standard technique for
molecular and cell biologists and provides ideal preservation of proteins for long periods when stored at − 80 °C [1]. Alternatively, formalin-fixed paraffin-embedded (FFPE) with proper sample preparation can increase proteome coverage and depth [2]. However, field experiments as in the
* R. R. Alyethodi [email protected]
A. K. De [email protected]
S. Karthik [email protected]
T. Sujatha [email protected]
K. Muniswamy [email protected]
Jai Sunder [email protected]
S. K. Ravi [email protected]
A. Kundu [email protected]
P. Perumal [email protected]
1
D. Bhattacharya [email protected]
ICAR-Central Island Agricultural Research Institute, Port Blair, Andaman & Nicobar Islands, India
P. A. Bala [email protected]
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case of animal experiments, remoteness of the sample collection site from the laboratory, and areas with insufficient infrastructure to allow quick access to liq
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