[Real Time PCR]#16 Effect of pre-analysis variables for circulating miRNA quantitation

관리자
22 Nov 2021
Views 32

Contents

1.Introduction 

2.Materials and equipment 

3. Procedure 

4. Results 

5. Precautions 

6. References 

1. Introduction


1. Introduction

There are not many studies on pre-analysis variables that affect the clinical usefulness of miRNA with sufficient biological potential. However, it was judged that it was important to find a more suitable blood sample for reliable results of circulating miRNA quantities in a clinical experimental environment. Therefore, we tested the effects of various environments such as additives, centrifugation speed, blood coagulation process, and cell contamination in the blood collection container   

2. Materials and equipment


2. Materials and equipment 

[blood collection container]

-ethylene diamine tetra-acetic acid (EDTA) tube

-serum separator tube(SST)

-sodium citrate tube

-plain tube


[Equipments]

-Centrifuge

-Hematology analyzer (sysmex XE-2100, Kyto, Japan )

-Conventional PCR machine (ICycler Thermal Cycler)

-Quantitative PCR Machine (ABI7500 real time PCR system, USA )


[Reagents]

-Separation reagent for the Peripheral blood mononuclear cells (Ficoll-Paque PLUS Sigma Aldrich, USA)

-miRNA isolation kit (mirVana PARIS kit, Thermo-Fisher Scientific Co. USA)

-IC (Caenorhabditis elegans miRNA 39, USA)

-reverse transcription kit (Taqman miRNA RT kit, miRNA-specific stem-loop primers USA)

-real time PCR kit (Taqman Universal PCR master mix-ABI, miRNA primer & hydrolysis probes-ABI, USA )


[Statistics Program]

PASW statistics 18.0 software 

3. Procedure


3. Procedure 

[sample collection]

1. Blood of healthy adults was collected to EDTA, sodium citrate, SST, and plain containers.

2. All samples are centrifuged under two conditions (1) 795 g/ 20 minutes and (2) 15,000 g/ 10 minutes in the refrigerator (4℃) and collected supernatant.

3. Centrifugated plasma and serum were collected into 2/3 upper and 1/3 lower layers, and the number of blood cells (WBC, RBC, and PLT) was measured, respectively. 

4. Arbitrarily separated mononuclear cells (serial dilution: 10,000, 1,000, 100, 50, 25, 12 WBCs in 400uL serum) was added to the serum that underwent additional high-speed centrifugation.

5. The blood in the sodium citrate, plain container was dispensed immediately after blood collection, and centrifuged after 5, 10, 20, 25, and 30 minutes passed. 


[miRNA 분리]

- Processing after add 50pmol/L cell-miR-39 to 400 uL plasma, serum

- Using 50uL elution solution 

 

[RT]

nuclease-free H2O4.5 uL
5X Taqman miRNA RT primer3 uL
100 mmol/L deoxynucleoside triphosphates1.5 uL
50 U/uL MultiScribe reserve transcriptase1 uL
eluted RNA5 uL
Total15 uL

-16℃, 30 min. > 42℃, 30 min. > 85℃, 5 min.


[quantitative PCR]

nuclease-free H2O7 uL
2X Taqman Universal PCR master mix10 uL
20X Taqman miRNA Assay primer1 uL
RT product2 uL
Total20 uL

- 95℃, 10min. > (40cycles) 95℃, 15 sec./ 60℃, 60 sec.


[analysis] 

Find △Ct in target and compare and analyze. 

- (△Ct value = target substracting the cel-miR-39 Ct value)

4. Results


4. Results

- As the 7-day sample storage time passed, the amount of change increased in plasma than serum and room temperature than refrigeration temperature. 

- As the number of arbitrarily added mononuclear cells increases, the amount of mi RNA also increases. 

- Plasma has a large range of changes in the upper/ lower layers, while little changes in serum 

- In plasma, the higher the centrifugation rate, the significantly smaller the amount of mi RNA, while there is no significant difference in serum 

- The amount of mi RNA tends to increase during the blood clotting process, but it is not a significant difference. 


5. Precautions


5. Precautions 

- When collecting plasma and serum supernatants, move from the surface of the sample to follow the pipette tip. 

6. References

6. References 

- Kirschner MB, Kao SC, Edelman JJ, et al. Haemolysis during sample preparation alters microRNA content of plasma. Plos one 2011; 6: e24145

- Validation of circulating miRNA biomarkers for predicting lymph node metastasis in gastric cancer, J Mol Diagn. 2013 




The original author's post (korean) ↗


1 0