Evaluation of different strategies for real-time RT-PCR expression analysis of corticotropin-releasing hormone and related proteins in human gestational tissues.

Author(s): Sehringer B, Zahradnik HP, Deppert WR, Simon M, Noethling C, Schaefer WR

Publication: Anal Bioanal Chem, 2005, Vol. 383, Page 768-75

PubMed ID: 16189679 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to determine if real-time quantitative RT-PCR (qRT-PCR) analysis is influenced by a delay in biospecimen preservation, specimen thawing, homogenization method, reverse transcription technique and quantitation method.

Conclusion of Paper

A cold ischemia time of 2 h at room temperature did not significantly alter levels of corticotrophin-releasing hormone (CRH) or cyclophilin compared to controls snap-frozen after less than 30 min. However, thawing a specimen prior to homogenization for 5 min at room temperature did significantly raise cycle threshold (Ct) values for both CRH and cyclophilin compared to controls that were still frozen (p<0.05). Omitting DNase pre-treatment also elevated Ct values for CRH (p<0.01). Although statistical significance was not assessed, the authors report the efficiency of the RT reaction was increased when samples were primed with random hexamers as opposed to oligo dT, and SuperScript II as opposed to OmniScript reverse transcriptase. Quantitation by the relative rather than the standard curve methods also affected results in a transcript- and tissue-dependent manner although statistical significance was not determined.

Studies

Study Purpose

The aims of this study were to determine if real-time qRT-PCR analysis of CRH, CRH binding protein (CRG-BP) and receptors (CRH-R1, CRH-R2) in placental and myometrium is affected by a room temperature delay (cold ischemia time), specimen thawing prior to homogenization, the method of homogenization, use or omission of DNase pre-treatment, priming method and enzyme used during reverse transcription, the qPCR quantification method. Biopsies from normal placenta and myometrium specimens were rinsed in Hank's balanced salt solution before being snap-frozen in liquid nitrogen and stored at -80 degrees C until analysis. The number of specimen analyzed ranged between 3 and 5 and was dependent on the variable investigated.

Summary of Findings:

Real-time qRT-PCR analysis of CRH and cyclophilin levels did not differ significantly after a cold ischemia time of 2 h compared to controls snap-frozen after less than 30 min at room temperature. Ct values for both CRH and cylcophilin were approximately 1 and 2 Ct values higher, respectively, among specimens thawed for 5 min before homogenization compared to controls that were still frozen (p<0.05). Although data was not shown, the authors report that RNA quality by gel electrophoresis was not affected by homogenization method. Omitting the DNase pretreatment step resulted in CRH levels that were approximately 10 Ct values lower than DNase-treated placental RNA (p<0.01), although it is unclear if other transcripts were investigated. The efficiency of RT reaction was affected by priming method in both placenta and myometrium, as samples primed using random hexamers generated cyclophilin levels that were approximately 2-3 Ct values lower than those primed with oligo dT (p<0.01). The authors also report a higher RT efficiency with SuperScript II compared to OmniScript reverse transcriptase although data was not shown. Although statistical significance was not assessed for relative quantitation methods, the authors report that the standard curve and comparative Ct methods produced comparable results for CRH-R1 and -R2 mRNA levels in placenta, while CRH mRNA levels were modestly higher with the comparative Ct method in placenta. Similarly, relative amounts of CRH and CRH-BP mRNA were also influenced by normalization method in placenta but not myometrium or choriodecidua, as calculated levels were higher for both transcripts when normalized to cyclophilin compared to total RNA, while the authors report comparable results for CRH-R1 and -R2 although data was not shown. Further, Ct values obtained for cyclophilin differed among tissue types, with significantly higher Ct values in placenta compared to myometrium and choriodecidua (p<0.05).

Biospecimens

Preservative Types

Frozen

Diagnoses:

Normal

Platform:

Analyte	Technology Platform
RNA	Spectrophotometry
RNA	Real-time qRT-PCR
RNA	Electrophoresis

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Biospecimen Acquisition	Biospecimen location	Placenta Myometrium Choriodecidua
Biospecimen Acquisition	Cold ischemia time	<30 min 60 min 120 min
Storage	Thaw duration	0 min (frozen) 5 min
Analyte Extraction and Purification	Tissue homogenization	Bead milling Potter homogenizer Rotor-stator Hammer
Analyte Extraction and Purification	Nucleic acid digestion	Untreated DNase I
Real-time qRT-PCR Specific	Priming method	Oligo dT Random hexamer SuperScript II RT OmniScript RT
Real-time qRT-PCR Specific	Targeted nucleic acid	CRH CRH-BP CRH-R1 CRH-R2 Cyclophilin
Real-time qRT-PCR Specific	Data handling	Standard curve method Comparative Ct method

You Recently Viewed

News and Announcements

Most Downloaded SOPs in 2024

New Articles on the GTEx Project are Now FREELY Available!

Just Published!

More...