Jan 31, 2022
Quantification of Isolated Circulating MicroRNAs using Qiagen LNA Panels
This protocol is a draft, published without a DOI.
- 1University of Toronto
Protocol Citation: Dakota Gustafson 2022. Quantification of Isolated Circulating MicroRNAs using Qiagen LNA Panels. protocols.io https://protocols.io/view/quantification-of-isolated-circulating-micrornas-u-b4huqt6w
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: January 31, 2022
Last Modified: January 31, 2022
Protocol Integer ID: 57620
Keywords: MicroRNA, Circulating, PCR, Qiagen, LNA, Quantification, Normalization
Abstract
A protocol for quantification of circulating microRNA using the Qiagen LNA Low Density Array MicroRNAs isolated from platelet-poor plasma. During the purification step samples are spiked with cel-miR-39 as a mean of technical normalization. Post-collected normalization is performed using the 'Global Mean Normalization' methodology.
Guidelines
This protocol is for use with the miRCURY SYBR® Green PCR Kit (cat. nos. 339345, 339346, 339347) on any real-time PCR cycler. This protocol is used for conducting real-time PCR using the following PCR panels in 96- or 384-well format. This protocol is optimized for detection of miRNA targets with any real-time cycler and conditions for fluorescence normalization. The amount of required ROX dye varies, depending on the instrument used.
Materials
5x miRCURY RT SYBR® Green Reaction BufferQiagenCatalog #339340
10x miRCURY RT Enzyme MixQiagenCatalog #339340
UniSp6 RNA Spike-in Template dried down QiagenCatalog #339340
Nuclease-Free Water Contributed by users
2x miRCURY SYBR® Green PCR Master Mix QiagenCatalog #339346 SYBR Green PCR BufferQiagenCatalog #339346 ROX™ Reference DyeQiagenCatalog #339346 miRCURY LNA miRNA Panels Qiagen
Protocol materials
5x miRCURY RT SYBR® Green Reaction BufferQiagenCatalog #339340
10x miRCURY RT Enzyme MixQiagenCatalog #339340
UniSp6 RNA Spike-in Template dried down QiagenCatalog #339340
Nuclease-Free Water
2x miRCURY SYBR® Green PCR Master Mix QiagenCatalog #339346
SYBR Green PCR BufferQiagenCatalog #339346
ROX™ Reference DyeQiagenCatalog #339346
miRCURY LNA miRNA Panels Qiagen
10x miRCURY RT Enzyme MixQiagenCatalog #339340
UniSp6 RNA Spike-in Template dried down QiagenCatalog #339340
Nuclease-Free Water
5x miRCURY RT SYBR® Green Reaction BufferQiagenCatalog #339340
Nuclease-Free Water
2x miRCURY SYBR® Green PCR Master Mix QiagenCatalog #339346
SYBR Green PCR BufferQiagenCatalog #339346
ROX™ Reference DyeQiagenCatalog #339346
miRCURY LNA miRNA Panels Qiagen
Before start
Thaw reagents on ice prior to beginning experiment.
Reverse Transcription
Reverse Transcription
1h 5m
1h 5m
Dilute each template RNA sample to 5 ng/µl using nuclease-free water.
Prepare the reverse transcription master mix using:5x miRCURY RT SYBR® Green Reaction BufferQiagenCatalog #339340 10x miRCURY RT Enzyme MixQiagenCatalog #339340 UniSp6 RNA Spike-in Template dried down QiagenCatalog #339340 Nuclease-Free Water Contributed by users .
Incubate for 01:00:00 at 42 °C , then 00:05:00 at 95 °C , and immediately cool to 4 °C .
1h 5m
6. Place the reverse-transcription reactions on ice and proceed directly with real-time PCR. Follow the recommendations for proper cDNA dilution provided in the protocol for the PCR Assay or Panel to be used.
Note: If you do not plan to use the cDNA immediately, store it undiluted at 2–8ºC for up to 4 days or at –30 to –15ºC for up to 5 weeks. We recommend storing synthesized cDNA in low-nucleic acid binding tubes or plates.
Quantitative, Real-Time PCR Using miRCURY LNA miRNA Custom PCR Panels
Quantitative, Real-Time PCR Using miRCURY LNA miRNA Custom PCR Panels
Dilute the cDNA 1:80 according to the table immediately before use. We do not recommend storing this 1:80 dilution of cDNA.
Prepare the master reaction mix according to the table using:
Nuclease-Free Water Contributed by users
2x miRCURY SYBR® Green PCR Master Mix QiagenCatalog #339346 SYBR Green PCR BufferQiagenCatalog #339346 ROX™ Reference DyeQiagenCatalog #339346 miRCURY LNA miRNA Panels Qiagen
Vortex the reaction mix thoroughly and dispense 10 µl per well into the PCR panel plate(s).
Note: The experiment can be paused at this point. Store the reactions protected from light at 2–8°C for up to 24 h.
Seal the plate. Carefully vortex it to dissolve the primers (optional). Briefly centrifuge the plate(s) at room temperature. Wait 5 min while the primers dissolve in the reaction mix.
Program the real-time cycler according to the table.
Note: Data acquisition should be performed during the annealing/extension step.
Perform the initial data analysis using the software supplied with your real-time PCR
Save data and export.
Data Analysis
Data Analysis
Obtain raw Cq values (Cp or CT, depending on PCR instrument) and verify the run quality (i.e., checking for values >35; depending on the threshold being used).
Perform data management according to Qiagen's website to facilitate import:
Import data and conduct analysis appropriate to the method used (i.e., normalization to reference gene or global mean normalization), and explore downstream analysis.