Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

Impact of different stabilization methods on RT-qPCR results using human lung tissue samples.

Author(s): Esteva-Socias M, Gómez-Romano F, Carrillo-Ávila JA, Sánchez-Navarro AL, Villena C

Publication: Sci Rep, 2020, Vol. 10, Page 3579

PubMed ID: 32108147 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the effects of preservation of lung tissue using RNAlater, snap-freezing, Optimal Cutting Tissue (OCT) compound, and formalin-fixation paraffin-embedding on RNA quality, RNA Integrity Number (RIN), and gene expression levels. The effects of thaw time at room temperature on gene expression levels was also examined. Lung tissue was obtained by surgical excision or organ donation from 20 participants with no history of chronic obstructive pulmonary disease, asthma, or obstructive sleep apnea and no bronchodilator or corticoid therapy. Matched tissue specimens were preserved by snap-freezing in liquid nitrogen and storage in a −80 °C freezer; snap-freezing in OCT in an isopentane bath and then storage in a −80 °C freezer; placement in RNAlater, incubation for 24 hours at 4 °C, and then storage at −80°C, or formalin fixation in 4% neutral buffered formalin at room temperature for at least 24 hours, dehydration in a tissue processor, and then paraffin embedding. For RNA extraction, RNAlater-preserved, snap-frozen, and snap-frozen OCT embedded specimens were disrupted using a Tissue Lyser and RNA was extracted using the miRNeasy Mini Kit. RNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissue after deparaffinization using the RNeasy FFPE Kit. RNA concentration and A260/280 were assessed using a Nanodrop spectrometer. RINs were assigned by Nano assay and a bioanalyzer. cDNA was transcribed using the SensiFAST cDNA Synthesis Kit. RNA integrity was evaluated through endpoint PCR analysis of six fragments of the Hypoxanthine Phosphoribosyltransferase 1 (HPRT1) housekeeping gene (73, 156, 257, 347, 400, and 436 bp) followed by separation of the amplification products by agarose gel electrophoresis.  RNA quality was also evaluated by real-time qRT-PCR amplification of the HPRT1, SNRPD3, and Jun housekeeping genes. To examine effects due to thaw time, gene expression levels were compared between RNAlater, snap-frozen, and frozen OCT specimens thawed at room temperature for 0, 1, 3, 6, 12, 30, or 45 min before RNA extraction.

   Summary of Findings:

   The 18S and 28S rRNA electropherogram peaks were sharply defined in RNA from RNAlater and OCT specimens, distinguishable but with a decreased signal due to slight degradation in RNA from snap-frozen specimens, and not detectable in RNA from FFPE specimens. Consistent with the observation of the electropherograms, lower average RINs were observed for snap-frozen and FFPE specimens (5.2 and 1.4, respectively) than RNAlater-preserved and OCT specimens (7.6 and 8.1, respectively). The 260/280 ratios were around 2.0 for all specimens with the exception of FFPE tissues (260/280 = 1.884). Amplification of the six different fragments (73-436 bp) from the HPRT1 gene analyzed by endpoint PCR was observed in RNA from RNAlater, snap-frozen, and OCT specimens but not in RNA from FFPE specimens. Real-time RT-PCR showed significantly higher CT values for all three genes in FFPE specimens compared to the other preservation methods (P<0.0001, all). Slightly lower CT values for Jun were found in snap-frozen compared to OCT specimens (P<0.005). CT values were unaffected by thaw time for any of the genes examined in RNAlater-preserved specimens, but increased with increasing thaw time in snap-frozen and OCT specimens beginning at 6 min for HPRT1 and after 12 min for SNRPD3 and Jun (P<0.01, all).

   Biospecimens

   • Tissue - Lung

   Preservative Types

   • Formalin
   • RNAlater
   • OCT
   • Frozen

   Diagnoses:

   • Normal

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	OCT RNAlater Snap frozen Formalin (buffered)
   RT-PCR Specific	Targeted nucleic acid	HPRT1
   Real-time qRT-PCR Specific	Targeted nucleic acid	HPRT1 SNRPD3 Jun
   Storage	Thaw duration	0 min 1 min 3 min 6 min 12 min 30 min 45 min

   View more