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Simultaneous extraction of mRNA and microRNA from whole blood stabilized in tempus tubes.

Author(s): Richards J, Unger ER, Rajeevan MS

Publication: BMC Res Notes, 2019, Vol. 12, Page 39

PubMed ID: 30658701 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of extraction kit type on the yield, purity, and integrity of mRNA and miRNA from Tempus Blood Tubes. Blood was collected from six healthy donors into Tempus Blood RNA Tubes and immediately shaken vigorously for 10 sec. Blood was then stored at room temperature for 2 h followed by freezing at -20°C. RNA was extracted from thawed blood from another 12 patients using the Tempus Spin RNA Extraction Kit, the Norgen Preserved Blood RNA Kit, and the MagMax for Stabilized Blood RNA Isolation Kit. RNA was also extracted using the MagMax protocol with and without the DNAse step and using the manual and semi-automated (MagMax Express 96) methods. RNA yield and purity were determined using a NanoDrop. RNA integrity and small RNA fraction were evaluated using a bioanalyzer. mRNA was reverse-transcribed using the PrimeScript RT Kit with gDNA eraser and real-time RT-PCR amplified using primers for β-actin (ACTB), matrix metallopeptidase 9 (MMPP), arginase 1 (ARG1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), interleukin-2 receptor alpha chain (CD25/IL2RA), hypoxanthine phosphoribosyltransferase 1 (HPRT1), forkhead box P3 (FOXP3), phosphoglycerate kinase 1 (PGK1), and peptidyl-prolyl cis–trans isomerase B (PPIB). miRNA was reverse-transcribed and real-time RT-PCR amplified using the All-in-One miRNA qRT-PCR Reagent Kit along with primers for miR-16-5p, miR-30b-5p, miR-133a-3p, miR-155, miR-191, miR-103, miR-223, miR-26b, and snRNA.

   Summary of Findings:

   RNA yields were highest when the Norgen Extraction Kit was used and lowest when the MagMax Kits was used (P=0.008) but the differences were small (12.04 µg for Norgen, 10.14 µg for Tempus, and 8.34 µg for MagMax). Similarly, there were small differences in purity as determined by OD260/280 and OD260/230 noted (P=0.027 and P<0.0001, respectively). RNA integrity as determined by mean RNA integrity number (RIN) and 28S rRNA to 18S rRNA ratio was highest when extraction was performed using Tempus (8.70 and 1.69, respectively) followed by Norgen (8.63 and 1.54, respectively) and MagMax (6.68 and 1.26 respectively). Although the differences between kits were significant (P<0.001), the authors state all three kits produced RNA suitable for downstream assays. Small RNA yields were comparable among the kits, but there were significant differences in miRNA yields and the miRNA percentages of small RNA (P=0.011 and P=0.014, respectively). The miRNA yields and percentages were highest when MagMax was used (39.3 ng and 11.18%, respectively) followed by Tempus (24.7 ng and 8.16%, respectively), and Norgen (17.9 ng and 5.17%). Differences in the Cq values for each of the mRNAs among the three kits were limited to less than 2-fold, but the MagMax kit produced much lower Cq values for most of the miRNAs evaluated (average 12-fold more than Tempus which averaged 3.3-fold more than Norgen).

   The authors report no effect of automation or the inclusion of the DNAse step on the total RNA yield, purity (OD260/280 or OD 260/230), RIN, 28S to 18S rRNA ratio, small RNA yield, or miRNA yield when extraction was with the MagMax Kit. Further, there was no effect of inclusion of a DNAse step or automation of the MagMax kit on the Cq values for mRNA or miRNA.

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • Other Preservative

   Diagnoses:

   • Normal

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	MagMax for Stabilized Blood RNA Isolation Kit MagMax for Stabilized Blood RNA Isolation Kit with automation (MagMax Express 96) Norgen Preserved Blood RNA Kit Tempus Spin RNA Extraction Kit
   Real-time qRT-PCR Specific	Targeted nucleic acid	snRNA miR-26b miR-223 miR-103 miR-191 miR-155 miR-133a-3p miR-30b-5p miR-16-5p Peptidyl-prolyl cis–trans isomerase B Phosphoglycerate kinase 1 Forkhead box P3 Hypoxanthine phosphoribosyltransferase 1 Interleukin-2 receptor alpha chain Glyceraldehyde 3-phosphate dehydrogenase Arginase 1 Matrix metallopeptidase 9 β-actin
   Analyte Extraction and Purification	Nucleic acid digestion	No DNase DNase added

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