CN-114807301-B - High-throughput automatic plasma small RNA library construction method and application thereof

CN114807301BCN 114807301 BCN114807301 BCN 114807301BCN-114807301-B

Abstract

The invention provides a high-throughput automatic plasma small RNA library construction method and application thereof, in particular to construction of a high-throughput automatic plasma small RNA library, and the high-throughput automatic plasma small RNA library construction method can be used for quantitatively researching the expression level of plasma small RNA and excavating potential biomarkers related to diseases. By applying the optimization method, library construction can be completed on 96 micro small RNA samples with initial quantity less than 5ng in one day, and data with good quality can be obtained after high-throughput sequencing. Through performance evaluation analysis, the technology provided by the invention has good cross-batch stability, has good distinguishing capability on inherent biological differences of small RNA expression levels of different biological samples, and can be used for small RNA library construction experiments of ultra-large queues.

Inventors

ZHENG YUANTING
YAO XINTONG
SUN SHANYUE
YANG JINGCHENG
REN LUYAO
WANG HAIYAN
HOU WANWAN
YU YING
SHI LEMING

Assignees

复旦大学
复旦大学

Dates

Publication Date: 20260421
Application Date: 20210827
Priority Date: 20210827

Claims (4)

1. A high throughput automated small RNA library construction method using NEXTFLEX ® small RNA-seq kit for library construction and using the apparatus of the method is a Sciclone ® NGSx automated liquid workstation, comprising the steps of: (1) Providing an isolated small RNA sample; (2) 3 'linker ligation is performed on the small RNAs in the small RNA sample, so that a 3' linker-bearing sample is obtained; (3) Removing redundant 3 'connectors from the sample with the connectors at 3'; (4) Deactivating the 3' connector with the excess removed; (5) Performing 5 'linker ligation on the sample obtained in the step (4), thereby obtaining a sample with a 5' linker; (6) Performing reverse transcription on the sample obtained in the step (5), thereby obtaining a cDNA sample with joints at two ends; (7) Purifying the sample obtained in the step (6); (8) Carrying out library amplification on the purified sample obtained in the step (7), thereby obtaining an amplified product; (9) Purifying and banking the amplified product to obtain a small RNA library; wherein the excess 3' connector removal Excess Adapter Removal comprises the steps of: (3-1) adding 1.05-1.5 times of a joint removal reagent Adapter Depletion Solution and 2 times of magnetic beads of the volume ratio of the final product of the step (2) into the product of the step (2) for mixing; (3-2) adding isopropanol with the volume ratio of 3 times of the final product of the step (2) into the product of the step (3-1), and reacting for 5 minutes after mixing; (3-3) placing the sample plate on a magnetic rack after 5 minutes, standing for 5 minutes, and removing supernatant after the solution is clarified; (3-4) placing the sample plate back to the working platform, adding 140-180 uL of 80% ethanol, reacting for 30 seconds, and removing the ethanol; (3-5) repeating the step (3-4), and standing for 2-3 minutes to completely volatilize the ethanol; (3-6) adding buffer Resuspension Buffer with the volume ratio of 1-1.2 times of the final product of the step (2) into the product of the step (3-5), blowing and uniformly mixing to re-suspend the magnetic beads, and incubating for 2 minutes at room temperature; (3-7) placing the sample plate on a magnetic rack, and reacting for 3 minutes to enable the magnetic beads to be adsorbed to the positions of the magnetic rings of the pipe wall; (3-8) collecting supernatant which is 1 time of the volume ratio of the product in the step (3-7) to the final product in the step (2) after the solution is clarified, and transferring the supernatant to a new sample plate; (3-9) repeating steps (3-1) to (3-7), wherein the buffer solution of step (3-6) is replaced by the non-nucleic acid water with the volume ratio of 0.5-0.7 times of the final product of step (2); (3-10) after the solution is clarified, collecting the supernatant into a new sample plate; Wherein 3 '4N-adenylated random linker reagent 3' 4N Adenylated Adapter used in the 3 'linker ligation step in step (2) is diluted 3-5 fold before use and/or 5' 4N-random linker reagent 5 '4N Adapter used in the 5' linker ligation step in step (5) is diluted 3-5 fold before use.
2. The method of claim 1, wherein the 3' linker ligation step is performed in step (2) for an incubation time of greater than 10 hours.
3. The method of claim 1, wherein the incubation environment in which step (2) 3' linker ligation, step (4) deactivates the excess 3' linker, step (5) ligates the 5' linker and/or step (6) reverse transcription is performed using any one of a temperature controlled and stable dry thermostat, a temperature circulator, a thermal cycler.
4. The method of claim 1, wherein the step (9) of purifying comprises the steps of: (9-1) adding magnetic beads with the volume ratio of 1-2 times into the amplified products of the library in the step (8), shaking and uniformly mixing, and removing supernatant; (9-2) adding 80% ethanol with the volume ratio of 6-9 times of the product of the step (8) into the product of the step (9-1) for cleaning; (9-3) adding the non-nucleic acid water with the volume ratio of 0.25-0.5 times of the product of the step (8) into the product of the step (9-2) to be uniformly mixed, so that the magnetic beads are kept stand after being resuspended; (9-4) collecting supernatant with the volume ratio of the product in the step (8) being 0.3-0.5 as a final product of the library construction.

Description

High-throughput automatic plasma small RNA library construction method and application thereof Technical Field The invention belongs to the technical field of biology, and particularly relates to a high-throughput automatic plasma small RNA library construction method and application thereof. Background Extracellular small RNA refers to RNA molecules with extracellular length less than 200 nucleotides, including miRNA, Y-RNA, circRNA, lncRNA, and the like. The expression level of the small RNA derived from the blood plasma is closely related to the physiological state of the disease, and the small RNA has the advantages of easy sampling, high stability, rapid quantification and the like, and becomes an important candidate source of clinical biomarkers. The small RNA sequencing technology (small RNA sequencing, small RNA-seq) is a sequence analysis method based on a high-throughput sequencing technology, and has become an important means for finding small RNA biomarkers due to the advantages of high accuracy, wide detection range and the like. Based on the large-scale clinical cohort of plasma samples, researchers can use small RNA-seq to comprehensively quantify small RNAs in plasma samples from patient and control groups, looking for inter-group differential small RNAs as potential biomarker candidates. Thus, high quality small RNA-seq data is a necessary prerequisite for biomarker mining. However, cross-batch stability has become a bottleneck for large-scale queuing of small RNA data generation. Similar to RNA-seq, small RNA-seq data also face the problem of systematic differences between batches, i.e., batch effects. Serious batch effects can lead to coverage of biological signals, and therefore, cross-batch quantitative stability is essential for small RNA-seq quantitative methods. However, the construction experimental procedure of the small RNA-seq library is complex, the library quality is extremely easy to be influenced by operation details, and the traditional manual operation is difficult to meet the construction requirement of a large-scale queue library due to the limitations of low flux, easy operation errors and the like. Therefore, there is an urgent need in the art to develop a high throughput automated method for constructing a small plasma RNA library, which improves experimental efficiency and batch-to-batch reproducibility, and reduces the possibility of operating errors in the experiment. Disclosure of Invention The invention aims to provide a high-throughput automatic plasma small RNA library construction method. The first aspect of the invention provides a high throughput automated small RNA library construction method usingThe small RNA-seq kit is subjected to library establishment, and comprises the following steps: (1) Providing an isolated small RNA sample; (2) 3 'linker ligation is performed on the small RNAs in the small RNA sample, so that a 3' linker-bearing sample is obtained; (3) Removing redundant 3 'joints from a sample with joints at 3', and adding a joint depletion solution (Adapter Depletion Solution), magnetic beads and isopropanol, wherein the mixing volume ratio of the reagent to the sample is 1.05-2:1-3:2.5-4:1; (4) Deactivating the 3' connector with the excess removed; (5) Performing 5 'linker ligation on the sample obtained in the step (4), thereby obtaining a sample with a 5' linker; (6) Performing reverse transcription on the sample obtained in the step (5), thereby obtaining a cDNA sample with joints at two ends; (7) Purifying the sample obtained in the step (6); (8) Carrying out library amplification on the purified sample obtained in the step (7), thereby obtaining an amplified product; (9) And (3) purifying and banking the amplified product to obtain a small RNA library. The second aspect of the invention provides an automatic small RNA library construction method, which usesThe small RNA-seq kit is subjected to library establishment, and comprises the following steps: (1) Providing an isolated small RNA sample; (2) Carrying out 3 'joint connection on the small RNA in the small RNA sample so as to obtain a sample with a joint at the 3' end; (3) Removing the unattached excess 3' linker from the sample obtained in step (2); (4) Deactivating the unattached excess 3' linker in the sample obtained in step (3); (5) Performing 5 'linker ligation on the sample obtained in the step (4), thereby obtaining a sample with a 5' linker; (6) Performing reverse transcription on the sample obtained in the step (5), thereby obtaining a cDNA sample with joints at two ends; (7) Purifying the sample obtained in the step (6); (8) Carrying out library amplification on the purified sample obtained in the step (7), thereby obtaining an amplified product; (9) Purifying the amplified product and establishing a library so as to obtain a small RNA library; The method is characterized in that the purification step (7) and/or the purification step (8) does not carry out the step of enrichi