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RNA Extracted from Formalin-Fixed, Paraffin-Embedded Renal Biopsy Biospecimens: An Evaluation of Alternative Extraction Kits and the Effects of Storage Time.

Author(s): Lu Y, Zhang M, Jiang Q, Yin R, Zhu P, Wang H, Liu Z, Zheng C

Publication: Biopreserv Biobank, 2017, Vol. 15(4), Page 396-398

PubMed ID: 28426236 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the yield, purity, and integrity of RNA obtained from FFPE renal biopsy specimens using two different extraction kits. Renal biopsies (<1.2mm by 10mm) from twelve patients with IgA nephropathy were fixed for 4-6 h, paraffin-embedded, and stored at room temperature in the dark for 5 years. RNA was extracted from a single 5 µm section using both the RecoverAll Total Nucleic Acid Isolation kit for FFPE and RNeasy FFPE kit. RNA yield was determined spectrophotometrically, RNA integrity was assessed by bioanalyzer, and 86 bp and 136 bp amplicons of 18S rRNA were analyzed by real-time PCR.

   Summary of Findings:

   Slightly but not significantly higher RNA yields were obtained when RNA was extracted using the RecoverAll kit than the RNeasy kit (261 vs. 159 ng/5 mm section). The mean RIN values and purity were comparable in specimens extracted using RecoverAll and RNeasy (2.3 versus 2.4 and A260/A280 of 1.54 versus 1.50) and most of the extracted RNA fragments were <200nt, regardless of extraction method used.  However, the CT values for both 18S rRNA amplicons were higher when RNA was extracted using RNeasy rather than RecoverAll (21.66 versus 22.71, P<0.03 for the 86 bp fragment and 24.42 versus 22.02, P<0.001for the 136 bp fragment). Further, the difference in the CT values for the 136 bp fragment and the 86 bp fragment was lower for specimens extracted using the RecoverAll kit than the RNeasy kit (0.36 versus 1.71, P=0.03), confirming that the RNA obtained using the RecoverAll kit was more intact.

   Biospecimens

   • Tissue - Kidney

   Preservative Types

   • Formalin

   Diagnoses:

   • Other diagnoses

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Spectrophotometry
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	RNeasy FFPE kit RecoverAll Total Nucleic Acid Isolation Kit
   Real-time qRT-PCR Specific	Technology platform	Bioanalyzer
   Real-time qRT-PCR Specific	Targeted nucleic acid	18S rRNA
   Real-time qRT-PCR Specific	Length of gene fragment	86 bp 136 bp

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2. Study Purpose

   This study investigated the effects of storage duration on the yield, purity, and integrity of RNA obtained from FFPE renal biopsy specimens. Renal biopsies (<1.2mm by 10mm) from 24 patients with IgA nephropathy were fixed for 4-6 h, paraffin-embedded, and stored at room temperature in the dark for 5 years (12 specimens) or 10 years (12 specimens) before RNA extraction. RNA was extracted from a single 5 µm section using the RecoverAll Total Nucleic Acid Isolation kit for FFPE. RNA yield was determined spectrophotometrically, RNA integrity was assessed by bioanalyzer, and amplification of SYNPO, CTNNB1, and 86 bp and 136 bp amplicons of 18S rRNA were analyzed by real-time PCR.

   Summary of Findings:

   Specimens stored for 10 years had lower yields than those stored for 5 years (10.9 ng/µL versus 12.7 ng/µL, P=0.01), but had comparable RIN values (2.4 versus 2.5), 18S rRNA CT values (17.3 versus 17.4), SYNPO CT values (35.97 versus 35.73), CTNNB1 CT values (35.10 versus 35.87) and A260/A280 (1.33, both).

   Biospecimens

   • Tissue - Kidney

   Preservative Types

   • Formalin

   Diagnoses:

   • Other diagnoses

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Real-time qRT-PCR
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Real-time qRT-PCR Specific	Targeted nucleic acid	18S rRNA SYNPO CTNNB1
   Storage	Time at room temperature	5 years 10 years

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