Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

Impact of thawing on reference gene expression stability in renal cell carcinoma samples.

Author(s): Ma Y, Dai H, Kong X, Wang L

Publication: Diagn Mol Pathol, 2012, Vol. 21, Page 157-63

PubMed ID: 22847160 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this paper was to determine if post-thaw storage of malignant and normal adjacent kidney specimens at room temperature adversely affects transcript levels of common reference genes. Case-matched malignant and normal adjacent tissue were collected from 16 patients diagnosed with renal cell carcinoma (RCC).  Four aliquots (50 mg each) were collected from each kidney, snap-frozen by unspecified means, and stored at -80°C for longer than 1 week.  Time at room temperature prior to snap-freezing was limited to 15 min or less. Tissue aliquots were either immediately placed in Trizol (0 min) or held at room temperature for 5, 15, or 30 min prior to extraction of total RNA by the Trizol method. RNA concentration was determined by Nanodrop spectrophotometry, and RNA integrity was evaluated by RNA integrity number (RIN) with a bioanalyzer. Samples included in the study were limited to tumors that had > 75% tumor cells, and 0 min controls that yielded RNA with a 260/280 ratio of 1.9-2.10, a 260/230 ratio of 1.95-2.30, and a RIN>7.5.  RNA was DNase treated prior to reverse transcription with the Primescript RT Kit. cDNA samples were stored at -20°C. Levels of the following reference genes were analyzed by qRT-PCR: ACTB, ALAS1, BM, GAPDH, HMBS, HPRT, PP1A, RPLP0, TBP, and TUBB. Statistically significant correlations were identified by Spearman rank tests. 

   Summary of Findings:

   Kidney specimens that were thawed at room temperature for 15 or 30 min exhibited significantly lower RINs than case-matched specimens placed in Trizol immediately (0 min) and those thawed for 5 min (p<0.01).  All ten reference genes evaluated displayed increased variation with advancing post-thaw storage at room temperature, as evidenced by progressive increases in the mean coefficient of variance compared to controls placed in Trizol immediately (0 min: 6.5% ± 1.5%; 5 min: 8.8% ± 2.2%; 15 min: 9.8% ± 2.9%; 30 min: 8.5% ± 2.1%; p<0.01 for all). Cp values of all reference genes examined, with the exception of B2M, differed significantly between malignant and normal adjacent specimens for at least two of the four thaw timepoints; although, all specimens regardless of diagnosis displayed an increase in variation with thaw time. A positive correlation between 2^{Cp values} and thaw time at room temperature was also observed (P <0.05), and linear regression analysis revealed a positive relationship between Cp values and thaw time at room temperature for all ten reference genes evaluated. 

   Normfinder and geNorm identified PPIA (geNorm M=0.82, 0.85, and 0.76), followed by TUBB, as the most stable reference genes during the room temperature thaw timecourse, while GAPDH (geNorm M=1.38, 1.44, 1.39) was identified to be the least stable of the ten genes examined. The geNorm program proposed the use of seven genes to be used in combination for normalization of timecourse specimens (ACTB, TUBB, RPLP0, HPRT1, PPIA, HMBS, ALAS1). When Cp values were normalized to the intact (0 min) sample using two stable genes (PPIA or ALAS1), the most unstable gene (GAPDH), or the seven genes identified by geNorm software, the expression ratios yielded errors that ranged between 5-40%. The authors observed the greatest deviations in expression ratios occurred when Cp values were not normalized to a reference gene (the intact 0 min control was used as a calibrator), followed by normalization to an unstable reference gene (GAPDH). Normalization to the seven reference genes identified by geNorm resulted in relative stable expression ratios across the thaw duration timecourse. 

   Biospecimens

   • Tissue - Kidney

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Normal Adjacent
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Thaw duration	0 min 5 min 15 min 30 min
   Real-time qRT-PCR Specific	Data handling	Normalized to combination of ACTB, ALAS1, BM, GAPDH, HMBS, HPRT, PP1A, RPLP0, TBP, and TUBB Normalized to GAPDH Normalized to ALAS1 Normalized to PPIA
   Real-time qRT-PCR Specific	Targeted nucleic acid	ACTB ALAS1 B2M GAPDH HMBS HPRT PPIA RPLP0 TBP TUBB

   View more