Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

Evaluation of RNA isolation methods for microRNA quantification in a range of clinical biofluids.

Author(s): Roest HP, IJzermans JNM, van der Laan LJW

Publication: BMC Biotechnol, 2021, Vol. 21, Page 48

PubMed ID: 34362351 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This paper compared recovery of spiked-in and endogenous microRNAs (miRNA, miR) that were extracted using four different kits from serum, bile, urine and liver graft perfusate specimens. Blood and urine were collected from 16 patients within the 24 h following liver transplantation surgery. Bile was collected from the same patients presumably during surgery (details not provided). During transplantation, grafts were flushed with ViaSpan followed by human serum albumin (HSA); the HAS flush was the perfusate. Urine was centrifuged for 5 minutes at 3200 g at 4°C and frozen at -20°C. Serum was obtained by centrifugation for 10 min at 800 g at 18 °C and frozen at -20°C.  RNA was extracted from serum, bile, perfusate and urine specimens of each patient using the (1) Qiagen miRNeasy Kit, (2) Qiazol in combination with dr. GenTLE, (3) precipitation, (4) Norgen Total RNA isolation kit, and (5) miRCURY RNA isolation kit -biofluids. Cel-miR-39 was added to all specimens during the lysis step. Heparinase I and cel-miR-54 were added to the extracted RNA during reverse transcription with Taqman microRNA Reverse Transcription Kit. cDNA was stored at -20°C until quantification of cel-miR-39 (all specimens), cel-miR-54 (all specimens), miR-21 (serum only), miR-122 (serum, perfusate and bile), miR-221(serum, perfusate and bile), miR-30e (urine), and miR-92a (urine) by real-time PCR.

   Summary of Findings:

   Heparinase I treatment improved miRNA amplification of heparin contaminated perfusate specimens following extraction with RNeasy or Qiazol with dr. GenTLE; however, this step was not necessary when miRNA was extracted with the Norgen kit. Nevertheless, heparinase I treatment was included in all additional experiments.  Recovery of cel-miR-39 that was spiked into serum, bile, perfusate, and urine specimens was superior following extraction with the RNAeasy kit as opposed to the other kits investigated. Importantly, compared to the RNeasy extraction from the same specimen type, a significantly lower amount of spiked in cel-miR-39 was amplified from serum when extraction was with the Norgen or miRCURY kit (45.8% and 36.1%, respectively; P<0.05, both); from bile when extraction was with  the Norgen kit (1.6%, P<0.01); from urine when extraction was with the Norgen, miRCURY, or Qiazol with dr. GenTLE precipitation (41.5%, 10.3%, and 32.5%, respectively; P<0.01, all); and from perfusate when extraction was with the Norgen, miRCURY and Qiazol with dr. GenTLE precipitation (10.8%, 22.2%, and 29.8%, respectively; P<0.01, all). Importantly, recovered levels of cel-miR-54 from urine, perfusate and serum specimen were comparable among all four extraction methods evaluated. However, cel-miR-54 levels in bile were much lower in one specimen after extraction with the miRCURY kit, which also was discolored. In a follow-up experiment miRNA was extracted from ten bile specimens using the miRCURY kit and four specimens yielded a discolored RNA sample (presumed to be due to the presence of biliverdin); these specimens also had lower quantified levels of cel-miR-54 and cel-miR-39 than matched specimens extracted using the other methods evaluated. The authors conclude that biliverdin co-isolated with miRNA in bile that undergoes extraction with the miRCURY kit, and that biliverdin interferes with PCR. In serum specimens, levels of miR-21 were highest when extraction was with the RNeasy kits, although levels of miR-122 and miR-222 were higher when extraction was with the miRCURY kit or Qiazol with dr. GenTLE precipitation compared to extraction with the RNeasy kit (significance not determined). Similarly, for urine the highest levels of miR-30e were observed when extraction was with the RNeasy kit, while extraction with the Norgen kit or Qiazol with dr. GenTLE precipitation led to higher (and much more variable) mean levels of miR-92a (significance not determined). In bile specimens, levels of miR-122 and miR-222 were comparable when extracted using the RNeasy Kit, miRCURY Kit, or Qiazol with dr. GenTLE precipitation methods, but were lower when extracted using the Norgen kit (significance not determined). Levels of both miR-222 and miR-122 were highest when extraction from perfusate was with RNeasy, but the difference between methods was small.  Heparinase I treatment improved real-time PCR amplification in heparin-contaminated perfusate specimens when miRNA was extracted using the RNeasy kit or Qiazol with dr. GenTLE precipitation method, but this step was not necessary when extraction was with the Norgen kit.

   Biospecimens

   • Bodily Fluid - Serum
   • Bodily Fluid - Urine
   • Bodily Fluid - Bile
   • Bodily Fluid - Other

   Preservative Types

   • Frozen

   Diagnoses:

   • Other diagnoses

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Biospecimen location	Serum Urine Bile Perfusate
   Analyte Extraction and Purification	Analyte isolation method	Qiagen miRNeasy Kit Qiazol in combination with dr. GenTLE, precipitation Norgen Total RNA isolation kit miRCURY RNA isolation kit -biofluids
   Real-time qRT-PCR Specific	Template modification	Heparinase treated Untreated
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-21 miR-221 miR-122 miR-30e miR-92a cel-miR-39 cel-miR-54 miR-222

   View more