[Real Time PCR]#19 miRNA detected in RNA separated from plasma

관리자
13 Dec 2021
Views 183

Contents

1.Introduction 

2.Materials and equipment 

3. Procedure 

4. Results 

5. Precautions 

1. Introduction


1. Introduction

Detecting miRNA from RNA isolated from plasma.

-micoRNA (miRNA) is naturally occurring noncoding RNA about 20 nucleotides. miRNA mediates the post-transcription gene regulation process and plays an important role in numerous biological processes, including differentiation and development, cell signaling and response to infection.  Through the expression of miRNA, it adds meaning as an effective substance for diagnosing many diseases in the body. Introducing an experiment on how to detect miRNA from RNA extracted from plasma  

2. Materials and equipment


2. Materials and equipment 

- Materials

① Plasma sample 

② RNA extraction kits (Serum miRNA purification kit,:Genolution Ltd.: South Korea) 

③ Poly A tailing kit (회사명; 나라이름)

④ RT kit(회사명; 나라이름)

⑤ Real time PCR kit (회사명; 나라이름


- Equipment

① Conventional PCR machine

② Real time PCR machine (CFX96-BioRad Laboratories Inc.)

③ Centrifuge 

④ Nano-drop(Thermo-Fisher Scientific, USA) 

⑤ Pipette(Eppendorf microliter pipette, Germany)

⑥ E-tube

⑦ 8 strip tube/cap

3. Procedure


3. Procedure 

1) Extract RNA from Plasma

① Put 200ul of plasma in a tube containing a lysis buffer(blue color) and vortex for 20 seconds. 

② After adding chloroform 200ul, vortex for 10 seconds

③ Check and confirm the layer separation after centrifuge at 13,000 rpm, 10min, 4℃, (e.g. blue color, white color and transparent layer from the bottom)

④ Separate from the transparent supernatant with pipette and transfer it into a new 1.5ml tube(each 200ul, 3 times). Don’t bring white layers. 

⑤ centrifuge at 13,000 rpm, 5min, 4℃, after additional inverting of 800ul of isopropanol into the supernatant.

⑥ remove supernatant leaving only pellets, Use a pipette to make sure the pellets don’t fall off. 

⑦ Wash with 70% Ethanol 1ml, spin down for 2 minutes, and remove supernatant without pellets. Use a pipette to make sure the pellets don’t fall off

⑧ Spin down to completely remove the remaining ethanol.

⑨ After adding 30 ul of RNase free water, melt the pellet.

⑩ After measured it’s amount, and store it at -20℃.


2) Poly A tailing

① Mixture is made according to the composition of Table 1 on a 1.5㎖ tube (using four types of RNA extracted from plasma).

Table 1. Composition of each components for mixture  

Table 1. Composition of each components for mixture

② Completely close the mixture lid and tap for 5 seconds or vortex to mix evenly, and the spin down.

③ After adding 18ul of each mixture to the 0.2㎖ PCR tube, add 2ul of the extracted RNA sample. Close the cap, tap it, mix it, and spin down.

④ Put the PCR tube in the conventional PCR equipment.

⑤ Performed PCR under the below conditions;

Cycling stepTemperatureTime
Incubation37℃60 min.
Inactivation65℃20 min,
Cooling10℃5 min,

⑥ Store the product for RT at 4℃


3) Reverse Transcription

① Make a mixture according to the composition of [Table 2] in 1.5㎖ tube

[Table 2]  Mixture of the components for the Reverse Transcription

② Close the mixture lid completely, taping for 5seconds, or vortex to mix uniformly, and spin down.

③ After adding 18ul of each mixture to a 0.2㎖ PCR tube, add 2ul of Poly A product. Close the cap, tap it mix it, and spin down.

④ Put PCR tube in conventional PCR equipment.

⑤ Perform PCR under the below conditions;

Cycling stepTemperatureTime
Incubation37℃20 min.
RTase inactivation95℃10 min.
Cooling10℃5 min.

⑥ Store at 4℃ the product for Real time PCR.


3) Real time PCR

① Make a mixture according to the composition of [Table 3] on a 1.5㎖ tube

(It makes three types of miRNA target and one type of internal control oligonucleotide mix respectively.)

[Table 3.] Mixture of the components for real time PCR

② Closure the mixture lid completely and tap or vortex for 5 seconds to mix uniformly, and then spin down..

③ After adding 18ul of each mixture to 0.2㎖ PCR tube, add 2 ul of each RT product, close cap , tap & mix and then spin down.

④ Put the PCR tubes into the Real time PCR equipment.

⑤ Perform PCR under the below conditions.

Cycling stepTemperatureTimeCycles
Pre-denaturation95℃600 s1
Denaturaiton95℃20 s45
Annealing & Extension68℃60 s
(+plate read)

  ⑥ Analyze the results

4. Results


4. Results

- Results Image

① Check each Ct value.

② the results of detecting three types of miRNA target and one type of internal control with RNA extracted from 4 types of plasma (by graph color) were shown as below figure.

5. Precautions


5. Precautions 

① Gloves shall be worn to prevent degradation during RNA extraction, and to reduce freezing and thawing of the sample after extraction, keep the sample as aliquot enough to use and take it out whenever using it. 

② when using the product in each process of Poly A and RT, be careful not to mislead the sample attached to the lid. 

③ The Poly A-treated products are unstable, so it is recommended to discard what you couldn’t use after using them on the same day.




The original author's post (korean) ↗ 

1 0