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Whole blood RNA quick lysate and application

A lysate and fast technology, which is applied in the field of blood RNA extraction, can solve the problems of unfavorable RNA extraction and time-consuming, and achieve the effect of shortened extraction time, high purity and good integrity

Pending Publication Date: 2020-02-18
杭州联科生物技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In other various cell lysis methods, it is necessary to lyse red blood cells first, and then lyse after obtaining white blood cells. This type of method is time-consuming in operation, which is not conducive to the extraction of RNA from a large number of samples.

Method used

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  • Whole blood RNA quick lysate and application
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  • Whole blood RNA quick lysate and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] A rapid whole blood RNA lysate preparation method, each 1000ml volume is composed of the following component mass / volume:

[0037] Guanidine Thiocyanate, 236.32g

[0038] Ammonium thiocyanate, 60.89g

[0039] Anhydrous sodium acetate, 4.102g

[0040] Glacial acetic acid, 14.25ml

[0041] Sodium Lauroyl Sarcosinate, 2.5g

[0042] Sodium chloride, 11.68g

[0043] Glycerin, 75ml

[0044] Water saturated phenol solution, 500ml

[0045] The rest is DEPC treated water.

[0046] Rapidly lyse and extract RNA from 4 parts of 500 μl human anticoagulated whole blood, including the following steps:

[0047] Step 1, Whole blood lysis: add 1ml of the rapid lysis solution to a 2ml centrifuge tube, then add 500μl fresh human anticoagulated whole blood, cover the tube cap, vortex for 30 seconds, and centrifuge at 13,000rpm for 10 minutes; divide into 4 parts Layers are in the same situation, take a photo, such as figure 1 Shown in left 1;

[0048] Step 2: Pass the RNA supernat...

Embodiment 2

[0056] A method for preparing whole blood RNA fast lysate, each 1000ml volume is composed of the following component mass / volume:

[0057] Guanidine Thiocyanate, 118.16g

[0058] Ammonium thiocyanate, 30.45g

[0059] Anhydrous sodium acetate, 8.20g

[0060] Glacial acetic acid, 21.5ml

[0061] Sodium Lauroyl Sarcosinate, 5g

[0062] Sodium chloride, 5.84g

[0063] Glycerin, 50ml

[0064] Water saturated phenol solution, 300ml

[0065] The rest is DEPC treated water.

[0066] A 500μl portion of human anticoagulated whole blood was quickly lysed and RNA was extracted, including the following steps:

[0067] Step 1, Whole blood lysis: Add 1ml of the rapid lysis solution to a 2ml centrifuge tube, then add 500μl fresh human anticoagulated whole blood, cover the tube cap, vortex for 30 seconds, and centrifuge at 13,000rpm for 10 minutes; Such as figure 1 Shown on the right 1;

[0068] Step 2: Pass the RNA supernatant through the purification column: add 400 μl absolute eth...

Embodiment 3

[0073] A method for preparing whole blood RNA fast lysate, each 1000ml volume is composed of the following component mass / volume:

[0074] Guanidine Thiocyanate, 236.32g

[0075] Ammonium thiocyanate, 152.24g

[0076] Anhydrous sodium acetate, 2.75g

[0077] Glacial acetic acid, 9.5ml

[0078] Sodium Lauroyl Sarcosinate, 2.5g

[0079] Sodium chloride, 5.84g

[0080] Glycerin, 75ml

[0081] Water saturated phenol solution, 500ml

[0082] The rest is DEPC treated water.

[0083] RNA extraction was performed on 3 parts of human anticoagulated whole blood stored at -80°C for one month, including the following steps:

[0084] Step 1, whole blood lysis: add 1ml of the rapid lysate to a 2ml centrifuge tube, then add 500μl of thawed human anticoagulated whole blood, cover the tube cap, vortex for 30 seconds, and centrifuge at 13,000rpm for 10 minutes;

[0085] Step 2: Pass the RNA supernatant through the purification column: add 400 μl absolute ethanol to a new 1.5ml centrifug...

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Abstract

The invention provides whole blood RNA quick lysate which does not need erythrocyte pyrolysis firstly, and an RNA extraction method. According to the method, the usage of toxic reagents is reduced toa large extent, RNA extraction steps are simplified, and RNA obtained through purification is good in integrity and high in purity and can be directly used for various molecular biology experiment ofRT-PCR, Northern blot, Dot blot and the like. Every 1000ml by volume of the whole blood RNA quick lysate disclosed by the invention consists of the following components by dosage of 0.2M-4M of guanidine thiocyanate, 0.2M-3M of ammonium thiocyanate, 0.01M-0.2M of anhydrous sodium acetate, 0.05M-0.3M of glacial acetic acid, 0.1%-2%(W / V) of sodium lauroyl sarcosine, 0.02M-0.3M of sodium chloride, 1%-15%(V / V) of glycerine, 10%-75% (V / V) of a water saturation phenol solution, and the balance DEPC treating water.

Description

technical field [0001] The invention relates to the technical field of blood RNA extraction, in particular to a rapid whole blood RNA lysate and its application. Background technique [0002] Due to its easy-to-obtain characteristics, blood has the highest proportion of use in clinical auxiliary examination methods. Blood tests are not only the main basis for diagnosing various blood diseases, but also can provide a lot of important information for the diagnosis and identification of other systemic diseases. RNA extraction is a basic content in molecular biology research and the basis for gene expression analysis. The rapid extraction of whole blood RNA saves the time of scientific researchers engaged in clinical research, reduces the probability of RNA degradation, and can accelerate the rapid diagnosis of certain diseases. [0003] According to the different adsorption media for extracting RNA, the main methods for blood RNA extraction include precipitation method, magne...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/101C12Q2527/125
Inventor 周志鹏
Owner 杭州联科生物技术股份有限公司
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