Method for detecting lncRNA (long noncoding RNA) in plasma

A medium-to-long-chain, non-coding technology, which is applied in the field of detection of long-chain non-coding in plasma, which can solve the problems of easy degradation of RNA, no extraction of lncRNA, low content of total RNA, etc., and achieve the effect of high content extraction

Inactive Publication Date: 2014-08-06
NINGBO UNIV
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  • Abstract
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Problems solved by technology

However, plasma contains mucopolysaccharides, mucin and other substances. In the process of extracting total RNA with the Trizol method, because the physical and chemical properties of polysaccharides are very similar to RNA, polysaccharides and RNA can be precipitated at the same time, resulting in polysaccharide-rich transparent gel. Precipitation, which is not conducive to subsequent cDNA synthesis and PCR detection
In addition, the content of total RNA in plasma is low, and it is difficult to extract and detect. Trizol method for extracting total RNA in plasma has complex operation steps, low yield, and RNA is easy to degrade
Up to now, there is still no perfect technical method for extracting and detecting lncRNA in plasma / serum

Method used

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  • Method for detecting lncRNA (long noncoding RNA) in plasma
  • Method for detecting lncRNA (long noncoding RNA) in plasma

Examples

Experimental program
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Effect test

Embodiment 1

[0024] A method for detecting lncRNA in plasma, the steps are as follows:

[0025] 1. After anticoagulation of whole blood with EDTA, centrifuge at 3000 rpm for 10 minutes at room temperature, take 300 μl of the upper plasma and transfer it to the first RNase-free centrifuge tube, add 900 μl of Trizol LS reagent, vortex for 10 seconds, and stand at room temperature for 5 minutes; at 4°C, Centrifuge at 12000rpm for 10 minutes. The sample is divided into two layers. The upper layer is a pink homogenized liquid, and the lower layer is a darker viscous impurity layer. Transfer the upper layer (about 1000 μl) to a second RNase-free centrifuge tube and discard the precipitate. , to remove impurities such as polysaccharides and mucins;

[0026] 2. Add 0.2ml of chloroform to the second RNase-free centrifuge tube, vortex for 10 seconds, let stand at room temperature for 5 minutes, then centrifuge at 12,000rpm for 15 minutes at 4°C, and absorb the upper aqueous phase (prefer less than e...

Embodiment 2

[0032] The method is basically the same as in Example 1, except that in step 9, the upstream and downstream primers for amplification are replaced by AA174084-specific amplification primers for RMRP-specific amplification primers, and the amplification curve and solution of the expression level of AA174084 in plasma are detected. From the amplification curve, it can be obtained that the Ct values ​​of the two plasma samples detected are 34.21 and 34.91 respectively; from the melting curve, it can be known that the curve is a narrow single peak, and it can be seen that the Ct values ​​of each plasma sample AA174084 was amplified specifically without the interference of primer dimers and heterogeneous bands.

Embodiment 3

[0034] The method is basically the same as in Example 1, except that in step 9, the upstream and downstream primers for amplification are replaced by BM709340-specific amplification primers for RMRP-specific amplification primers, and the amplification curve and solution of the expression level of BM709340 in plasma are detected. From the amplification curve, it can be obtained that the Ct values ​​of the two plasma samples detected are 27.87 and 30.46 respectively; from the melting curve, it can be known that the curve is a narrow single peak, and it can be seen that the Ct values ​​of each plasma sample are BM709340 was amplified specifically without the interference of primer dimers and heterogeneous bands.

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Abstract

The invention discloses a method for detecting lncRNA in plasma. Through steps of TrizolLS pretreatment of theplasma, chloroform and isoamyl alcohol mixed liquid extraction, isopropanol precipitation, ethanol washing, RNA RT (reverse transcription), PCR (polymerase chain reaction) detection and the like, a TrizolLSreagentcan well remove impurities such as polysaccharide, mucoprotein and the like in blood, RNA degradation is less, the phenomenon thattrichloromethane is easy to emulsify under the TrizolLS strong acid condition is effectively eliminated by a chloroform and isoamyl alcohol mixed liquid, high-quality and high-content extraction of RNA in the plasma is realized, fluorescence quantitative RT-PCR detection is performed on the quality of lncRNA, and meanwhile, a reference Ct (cycle threshold) value of the total RNA (that is, cDNA) mass can be detected.

Description

technical field [0001] The invention relates to a method for extracting and detecting RNA in plasma, in particular to a method for detecting long-chain noncoding in plasma. Background technique [0002] With the widespread application of high-throughput whole-genome sequencing technology, it is surprising to find that about 98% of transcripts in mammalian cells are non-coding RNA molecules (noncoding RNA, ncRNA). These non-coding RNA molecules include the well-known housekeeping (housekeeping) non-coding RNAs (such as tRNA and rRNA, etc.), small non-coding RNAs (such as microRNA and piRNA, etc.), and more are yet to be further studied. The long noncoding RNA (lncRNA) molecules studied. [0003] LncRNA refers to a class of RNA molecules whose transcripts are longer than 200 nucleotides (nucleotide, nt) and do not have the function of encoding proteins. Such molecules generally have highly conserved sequence elements, specific spatial secondary structures, cleavage forms and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6806C12Q1/6851C12Q2523/113C12Q2531/113
Inventor 郭俊明邵永富肖丙秀
Owner NINGBO UNIV
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