TITLE: Quantification of Isolated Circulating MicroRNAs using Qiagen LNA Panels
AUTHORS: Dakota  Gustafson

[DESCRIPTION]
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. 

[BEFORE_START]
Thaw reagents on ice prior to beginning experiment. 

[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.

[STEPS]
SECTION: Reverse Transcription
1. Dilute each template RNA sample to 5 ng/µl using nuclease-free water.
SECTION: Reverse Transcription
2. Prepare the reverse transcription master mix using:       .
SECTION: Reverse Transcription
3.   

SECTION: Reverse Transcription
4. Incubate for 60 min  at 42 °C , then 5 min  at 95 °C , and immediately cool to 4 °C .

SECTION: Reverse Transcription
5. 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.
SECTION: Quantitative, Real-Time PCR Using miRCURY LNA miRNA Custom PCR Panels
6. Dilute the cDNA 1:80 according to the table immediately before use. We do not recommend storing this 1:80 dilution of cDNA.

  

SECTION: Quantitative, Real-Time PCR Using miRCURY LNA miRNA Custom PCR Panels
7. Prepare the master reaction mix according to the table using: 
  
        


  

SECTION: Quantitative, Real-Time PCR Using miRCURY LNA miRNA Custom PCR Panels
8. 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.
SECTION: Quantitative, Real-Time PCR Using miRCURY LNA miRNA Custom PCR Panels
9. 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.
SECTION: Quantitative, Real-Time PCR Using miRCURY LNA miRNA Custom PCR Panels
10. 
Program the real-time cycler according to the table.
Note: Data acquisition should be performed during the annealing/extension step.

  


SECTION: Quantitative, Real-Time PCR Using miRCURY LNA miRNA Custom PCR Panels
12. Save data and export. 
SECTION: Quantitative, Real-Time PCR Using miRCURY LNA miRNA Custom PCR Panels
11. Perform the initial data analysis using the software supplied with your real-time PCR
SECTION: Data Analysis
13. 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). 
SECTION: Data Analysis
14. Perform data management according to Qiagen's website to facilitate import: 

https://geneglobe.qiagen.com/us/analyze
SECTION: Data Analysis
15. Import data and conduct analysis appropriate to the method used (i.e., normalization to reference gene or global mean normalization), and explore downstream analysis.