Optimizing RNA Extraction Methods for High Throughput Transcriptome Sequencing of Fresh Frozen and Formalin-Fixed Paraffin-Embedded Cardiac Tissues Specimens
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ABSTRACT:
PROVIDER: PRJNA1112367 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR29058073_1.fastq.gz | Fastqsanger.gz | |||
| SRR29058073_2.fastq.gz | Fastqsanger.gz | |||
| SRR29058074_1.fastq.gz | Fastqsanger.gz | |||
| SRR29058074_2.fastq.gz | Fastqsanger.gz | |||
| SRR29058075_1.fastq.gz | Fastqsanger.gz |
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Project description:We determined the best extraction buffer for proteomic investigation using formalin-fixation and paraffin-embedded (FFPE) specimens. A Zwittergent 3-16 based buffer, sodium dodecyl sulfate (SDS)-containing buffer with/without polyethylene glycol 20000 (PEG20000), urea-containing buffer, and FFPE-FASP protein preparation kit were compared for protein extraction from different types of rat FFPE tissues, including the heart, brain, liver, lung, and kidney. All of the samples were divided into two groups of laser microdissected (LMD) and non-LMD specimens. For both kinds of specimens, Zwittergent was the most efficient buffer for identifying peptides and proteins, was broadly applicable to different tissues without impairing the enzymatic digestion, and was well compatible with mass spectrometry analysis. As a high molecular weight carrier substance, PEG20000 improved the identification of peptides and proteins; however, such an advantage is limited to tissues containing submicrograms to micrograms of protein. Considering its low lytic strength, urea-containing buffer would not be the first alternative for protein recovery. In conclusion, Zwittergent 3-16 is an effective buffer for extracting proteins from FFPE specimens for downstream proteomics analysis.
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