fulltext- http://www.biomedcentral.com/content/pdf/1756-0500-4-324.pdf
Identification of Phosphoglycerate Kinase 1 (PGK1) as a Reference Gene
for Quantitative Gene Expression Measurements in Human Blood RNA
Virginia R Falkenberg, Toni Whistler, Janna' R Murray, Elizabeth R
Unger and Mangalathu S Rajeevan
BMC Research Notes 2011, 4:324
Published: 6 September 2011
Abstract (provisional)
Background
Blood is a convenient sample and increasingly used for quantitative
gene expression measurements with a variety of diseases including
chronic fatigue syndrome (CFS). Quantitative gene expression
measurements require normalization of target genes to reference genes
that are stable and independent from variables being tested in the
experiment. Because there are no genes that are useful for all
situations, reference gene selection is an essential step to any
quantitative reverse transcription-PCR protocol. Many publications
have described appropriate genes for a wide variety of tissues and
experimental conditions, however, reference genes that may be suitable
for the analysis of CFS, or human blood RNA derived from whole blood
as well as isolated peripheral blood mononuclear cells (PBMCs), have
not been described.
Findings
Literature review and analyses of our unpublished microarray data were
used to narrow down the pool of candidate reference genes to six. We
assayed whole blood RNA from Tempus tubes and cell preparation tube
(CPT)-collected PBMC RNA from 46 subjects, and used the geNorm and
NormFinder algorithms to select the most stable reference genes.
Phosphoglycerate kinase 1 (PGK1) was one of the optimal normalization
genes for both whole blood and PBMC RNA, however, additional genes
differed for the two sample types; Ribosomal protein large, P0 (RPLP0)
for PBMC RNA and Peptidylprolyl isomerase B (PPIB) for whole blood
RNA. We also show that the use of a single reference gene is
sufficient for normalization when the most stable candidates are used.
Conclusions
We have identified PGK1 as a stable reference gene for use with whole
blood RNA and RNA derived from PBMC. When stable genes are selected it
is possible to use a single gene for normalization rather than two or
three. Optimal normalization will improve the ability of results from
PBMC RNA to be compared with those from whole blood RNA and
potentially allows comparison of gene expression results from blood
RNA collected and processed by different methods with the intention of
biomarker discovery. Results of this study should facilitate
large-scale molecular epidemiologic studies using blood RNA as the
target of quantitative gene expression measurements.
---------------------------------------------
Send posts to CO-CURE@listserv.nodak.edu
Unsubscribe at http://www.co-cure.org/unsub.htm
---------------------------------------------
Co-Cure's purpose is to provide information from across the spectrum of
opinion concerning medical, research and political aspects of ME/CFS and/or
FMS. We take no position on the validity of any specific scientific or
political opinion expressed in Co-Cure posts, and we urge readers to
research the various opinions available before assuming any one
interpretation is definitive. The Co-Cure website <www.co-cure.org> has a
link to our complete archive of posts as well as articles of central
importance to the issues of our community.
---------------------------------------------
