PTEN transcript variants caused by illegitimate splicing in "aged" blood samples and EBV-transformed cell lines.

Author(s): Liu Y, Malaviarachchi P, Beggs M, Emanuel PD

Publication: Hum Genet, 2010, Vol. 128, Page 609-14

PubMed ID: 20839010 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of delayed isolation of MNC before nucleic acid extraction on RNA quality, detection of RNA SV, and mutations in PTEN. RNA and DNA were extracted using Trizol, and DNA was removed from RNA with the DNA-free kit. Full length PTEN was amplified using RT-PCR primers in the 3' and 5' untranslated regions.

   Summary of Findings:

   When blood was stored for 48 h at room temperature before isolation of MNC and extraction of RNA, RINs were significantly lower than those observed when MNC were isolated from blood immediately after collection (9.3 versus 8.05, p<0.01). Full length amplification of PTEN transcripts was possible in all specimens. Seven SV were found only in the 20 specimens stored at room temperature for 48 h before isolation of MNC and none were found in the corresponding specimens processed immediately. The authors believe these splice variants are unlikely to affect protein levels, as they are present in Epstein-Barr virus transformed lymphoblastoid cell lines where they do not affect PTEN protein levels. The authors report that no mutations in PTEN genomic sequences were identified, regardless of delayed isolation.

   Biospecimens

   • Bodily Fluid - Blood
   • Cell - Blood

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	DNA sequencing
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	RT-PCR
   RNA	Electrophoresis

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Time at room temperature	0 h 48 h

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