CN109593835B - Method, kit and application for evaluating trace FFPE RNA sample - Google Patents
Method, kit and application for evaluating trace FFPE RNA sample Download PDFInfo
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Abstract
The application discloses a method, a kit and application for evaluating trace FFPE RNA samples. The application relates to a method for evaluating trace FFPE RNA samples, which comprises the steps of selecting target fragments, and carrying out real-time fluorescence PCR on trace FFPE RNA samples by adopting specific primers and probes of the target fragments; and carrying out relative quantification by adopting a standard curve to obtain the relative concentration and abundance of the target fragment, thereby evaluating the usability of the trace FFPE RNA sample. The method breaks the limitation of the current mainstream detection tool 2100Bioanalyzer, adopts real-time fluorescence PCR and a standard curve to carry out relative quantification, judges the sample quality, and can directly display the abundance and usability of the target fragment compared with the 2100Bioanalyzer method, and the method does not need to additionally purchase expensive capillary electrophoresis analysis equipment, has lower cost and is easier to popularize and popularize.
Description
Technical Field
The application relates to the field of nucleic acid sample quality detection, in particular to a method, a kit and application for detecting the quality of a trace FFPE RNA sample.
Background
With the rapid development of next generation sequencing technology NGS, transcriptome sequencing technology has become a major means of studying gene expression. Transcriptomes are the necessary tie linking genomic genetic information with biological functions, and are the basis and starting point for gene function and structure studies. Through the new generation high throughput sequencing, almost all transcript sequence information of a specific tissue or organ of a certain species in a specific state can be comprehensively and rapidly obtained, and the method is widely applied to the fields of basic research, clinical diagnosis, drug research and the like.
In disease research and pathological analysis, after a fresh tissue sample obtained by means of surgery, puncture, endoscopic forceps and the like leaves a body, the fresh tissue sample is prepared into a paraffin block through the steps of fixation, dehydration, paraffin embedding and the like, and the paraffin block is called a Formalin-Fixed and Paraffin-Embedded (FFPE) sample. The FFPE sample is subjected to steps of slicing, staining and the like for pathological morphological diagnosis. The prepared FFPE sample can be stored for a long time under the room temperature condition due to stable property, is the most effective means for archiving medical records and diagnosing evidences of diseases, and is also a main material source for disease related analysis. Molecular biological detection by adopting FFPE samples is a main means for assisting diagnosis and realizing personalized medical treatment. However, the quality of the RNA samples extracted from FFPE samples, i.e., FFPE RNA samples, generally cannot meet the basic requirements of transcriptome sequencing experiments. Therefore, a higher requirement is put on the quality of the FFPE RNA sample to be detected rapidly, accurately and in high flux.
Prostate cancer is the second most common cancer worldwide, and is also the fifth leading cause of death in men associated with cancer. Prostate cancer is the most common cancer in men in 84 countries and is more common in developed countries, but the prevalence in developing countries is rising. Studies have shown that men over 60 years old who do not die of prostate cancer, about 30% to 70% of which have had prostate cancer. The transcriptome research for the prostate cancer has a great demand, and a mature and accurate FFPE RNA sample quality detection method is urgently needed, so that a large amount of precious clinical slice samples are promoted to be transformed to enter downstream molecular biology research.
RIN (RNA Integrity Number) is a tool developed by Agilent Technologies for indicating the quality of an RNA sample. RIN was applied to 2100Bioanalyzer, whose computational model was modeled from data from nearly 1300 human/rat/mouse samples. The calculation parameters of RIN are obtained from the complete track of RNA sample electrophoresis, so that the accuracy and the repeatability of the RIN are higher than those of the traditional quality control methods such as 28S/18S; also, because RIN is a single absolute value, interpretation of sample quality from person to person can be avoided. RIN is considered a gold standard for detecting the integrity of RNA samples, however its use still has a number of limitations.
1) RIN has a relatively high detection concentration threshold, and even if the high-sensitivity Agilent RNA 6000 Pico Kit is used, the concentration of a sample to be detected is required to reach more than 250 pg/mu L, which is a huge constraint factor for realizing molecular biology application of a large number of precious clinical samples;
2) RIN cannot directly predict the accuracy of the quantitative gene test results. Degradation of RNA is a gradual, heterogeneous process, so RIN does not represent the complete condition of mRNA. Different researchers or experimental designs will also have different requirements for RIN; if the Illumina official suggests, in the transcriptome experiment, only RNA samples with RIN more than or equal to 8 are selected; there are other groups of research that samples with RIN.gtoreq.6 can be considered to be of high quality during sample collection. Such simple cut-off, lack of adequate knowledge of the sample pretreatment method and experimental design, can lead to deviations in experimental results and expectations.
3) RIN does not substantially provide an effective indication of FFPE RNA sample quality. Since FFPE RNA samples are often highly fragmented, RIN is not sensitive to feedback on the extent of RNA degradation, and thus the success rate of library construction and the extent of data confidence of the detected samples cannot be predicted. Also, 18S and 28S are not generally visible due to the high degradation of FFPE RNA samples, which also results in the reference value of RIN becoming limited.
4) RIN has a strict applicable concentration range, clinical specimens are often at a premium, and often the RNA extracted does not meet the minimum concentration requirements of 2100 Bioanalyzer.
Disclosure of Invention
The application aims to provide a novel method, a kit and application for detecting the quality of trace FFPE RNA samples.
The application adopts the following technical scheme:
one aspect of the application discloses a method for evaluating trace FFPE RNA samples, which comprises the steps of selecting target fragments, and carrying out real-time fluorescence PCR detection on the trace FFPE RNA samples to be detected by adopting specific primers and probes of the target fragments; and analyzing the real-time fluorescence PCR detection result by adopting a standard curve to obtain the relative concentration and relative abundance of the target fragment in the trace FFPE RNA sample, thereby evaluating the usability of the trace FFPE RNA sample.
The availability refers to whether a trace FFPE RNA sample can be used for subsequent RNA sequencing analysis, and the purpose of the trace FFPE RNA sample evaluation is to judge whether the trace sample is suitable for RNA sequencing or not, so that the unqualified trace FFPE RNA sample is prevented from entering an RNA sequencing process, and unnecessary loss and waste of manpower and material resources are caused. On the one hand, judging whether the trace FFPE RNA sample can reach the nucleic acid amount of RNA sequencing according to the measured relative concentration and relative abundance; on the other hand, the real-time fluorescence PCR also shows the sequencing PCR process, and intuitively shows the feasibility of RNA sequencing, because the RNA fragments with high fragmentation or molecular damage cannot be reversely transcribed and amplified to generate fluorescent signals, the traditional Qubit spectrophotometry or RIN-based Agilent 2100 method cannot detect the quantity and purity of the sample, and the fragmentation or molecular damage of the sample cannot be detected. It can be understood that the real-time fluorescence PCR and the RNA sequencing are both PCR amplification processes, so if the evaluation method of the application can obtain better results, the evaluation method proves that the PCR can be smoothly performed, namely the fragmentation degree or the molecular damage degree can be reflected, and the RNA sequencing can be smoothly performed under the condition that the determined amount of the sample relative quantification can meet the requirement of the RNA sequencing, namely the effective RNA sequencing result can be obtained. The Qubit spectrophotometry or Agilent 2100 method can only detect and evaluate the amount and purity of the sample, and can not show the PCR amplification process, and can not detect the fragmentation degree or the molecular damage degree; therefore, even if a sufficient amount of sample is detected by the Qubit spectrophotometry or Agilent 2100 method, smooth RNA sequencing cannot be ensured.
The selection of the target fragment depends on a specific detection object or an object to be evaluated, for example, in one implementation of the present application, the quality evaluation is performed on a trace FFPE RNA sample of the prostate cancer, and the target fragment is a gene fragment representative of the prostate cancer. It should be noted that, the method for evaluating trace FFPE RNA samples only evaluates the relative concentration and relative abundance of the target fragment in the trace FFPE RNA samples, and the disease represented by the target fragment or the disease and related information thereof can be detected and obtained, and further detailed and accurate information can be obtained through subsequent RNA sequencing. For example, in one implementation of the application, the quality of a trace FFPE RNA sample of prostate cancer is assessed by detecting two key genes, GUSB gene and CDKN1A gene, of prostate cancer; however, whether these two key genes have pathogenic mutations or not, whether they cause prostate cancer, and RNA sequencing is also required for knowledge. The method of the application is only responsible for evaluating the quality of trace FFPE RNA samples.
It can be understood that the method for evaluating trace FFPE RNA samples finally tests the quality of the target fragment of the disease to be tested; specific primers and probes are designed for their target fragments or key genes, and re-evaluated according to the methods of the application, relative to other diseases.
It should also be noted that when FFPE RNA is used as an experimental material for RNA sequencing, there are two technical difficulties: ffpe treatment can lead to high fragmentation of RNA, and highly fragmented RNA fragments cannot participate in downstream pooling; ffpe treatment results in RNA being chemically modified at random sites, which results in reduced efficiency of reverse transcriptase or PCR enzymes when used as templates, resulting in library construction failure or loss of information carrying important biological significance. Existing methods for detecting or evaluating nucleic acid samples, including Qubit spectrophotometry and RIN-based Agilent 2100 methods, are not capable of exhibiting or predicting the degree of nucleic acid fragmentation or molecular damage of a sample, except for deviations in the accuracy of quantitative results. The method provided by the application provides a rapid and stable technical scheme by carrying out relative quantitative detection on target fragments aiming at the experimental method and the research purpose of FFPE RNA sequencing application. The relative quantitative result of the target fragment is not only the evaluation of molecular weight, but also the fragmentation and molecular damage degree of the RNA sample to be detected are judged through an RT-qPCR experiment, the RNA fragment with high fragmentation or molecular damage cannot be reversely transcribed and amplified to generate a fluorescent signal, the possibility that a trace FFPE RNA sample can be used for a downstream NGS experiment is shown, and a sample availability evaluation scheme which is specific to the trace FFPE RNA sample and takes experimental data as a guide is provided.
Preferably, the standard curve is obtained from an RNA standard using the same real-time fluorescent PCR amplification as the trace FFPE RNA sample.
Preferably, the RNA standard is a UHRR human standard.
The standard curve of the real-time fluorescent PCR is obtained by a conventional standard curve construction method, for example, the standard is subjected to gradient dilution and then real-time fluorescent PCR, thereby obtaining the standard curve, which is not particularly limited herein.
The other side of the application discloses the application of the method in the quality evaluation of trace FFPE RNA samples of the prostate cancer.
It can be understood that the method for evaluating trace FFPE RNA samples can be suitable for evaluating and detecting RNA samples of various cancers, and only needs to select target fragments or key genes related to the cancers and design corresponding specific primers and probes for real-time fluorescence PCR detection.
The application also discloses a method for evaluating trace FFPE RNA samples of the prostate cancer, which comprises the steps of selecting key genes of the prostate cancer, and carrying out real-time fluorescence PCR detection on the trace FFPE RNA samples to be tested by adopting specific primers and probes of the key genes; and analyzing the real-time fluorescence PCR detection result by adopting a standard curve to obtain the relative concentration and relative abundance of the prostate cancer key genes in the trace FFPE RNA sample, thereby evaluating the usability of the trace FFPE RNA sample.
Preferably, the key genes of prostate cancer include GUSB gene and CDKN1A gene, and real-time fluorescent PCR is detected as multiplex real-time fluorescent PCR of both genes.
The GUSB and CDKN1A genes have strong correlation with prostate carcinogenesis, and are often used as main target genes for prostate cancer transcriptome sequencing research, so the gene is used as a key gene of prostate cancer. It will be appreciated that other key genes may be selected, not only the GUSB gene and the CDKN1A gene, depending on the purpose of the study or in other circumstances.
Preferably, the upstream primer and the downstream primer of the specific primer of the GUSB gene are respectively a sequence shown by a Seq ID No.1 and a sequence shown by a Seq ID No.2, and the specific probe of the GUSB gene is a sequence shown by a Seq ID No. 3; the upstream primer and the downstream primer of the specific primer of the CDKN1A gene are respectively a sequence shown by a Seq ID No.4 and a sequence shown by a Seq ID No.5, and the specific probe of the CDKN1A gene is a sequence shown by a Seq ID No. 6;
Seq ID No.1：5’-TGATCGCTCACACCAAATCC-3’
Seq ID No.2：5’-CCTTGTCTGCTGCATAGTTAGAGTTG-3’
Seq ID No.3：5’-FAM-CTCCCGGCCTGTGACCTTTGTGA-TAMRA-3’
Seq ID No.4：5’-CAAACACCTTCCAGCTCCTGTAA-3’
Seq ID No.5：5’-AACGGGAACCAGGACACATG-3’
Seq ID No.6：5’-VIC-ATACTGGCCTGGACTGTTTTCTCTCGGC-TAMRA-3’。
the primers and the probes are designed specifically for the multiplex real-time fluorescent PCR detection of the GUSB gene and the CDKN1A gene, the two groups of primers and the probes can be matched with each other to be used as a whole body of organic combination for carrying out multiplex real-time fluorescent PCR, the simultaneous detection of the GUSB gene and the CDKN1A gene is realized, and the two groups of primers and the probes can not interfere with each other to influence the respective amplification efficiency or generate non-specific amplification.
Preferably, the standard curve is obtained from an RNA standard using real-time fluorescent PCR amplification of the same trace FFPE RNA samples.
Preferably, the RNA standard is a UHRR human standard.
The application also discloses a kit for evaluating trace FFPE RNA samples of the prostate cancer, which comprises specific primers and probes for detecting GUSB genes and CDKN1A genes, wherein the upstream and downstream primers of the specific primers of the GUSB genes are respectively a sequence shown by a Seq ID No.1 and a sequence shown by a Seq ID No.2, and the specific probes of the GUSB genes are sequences shown by a Seq ID No. 3; the upstream and downstream primers of the specific primer of the CDKN1A gene are respectively a sequence shown by a Seq ID No.4 and a sequence shown by a Seq ID No.5, and the specific probe of the CDKN1A gene is a sequence shown by a Seq ID No. 6.
It should be noted that, the trace FFPE RNA sample evaluation of prostate cancer of the present application realizes the two sets of primers and probes for efficient amplification according to the present application, so that for convenience of use, the present application uses them as a kit alone; of course, other reagents required for real-time fluorescent PCR may be contained in the kit, and are not particularly limited herein.
The application has the beneficial effects that:
according to the method for evaluating the trace FFPE RNA sample, the real-time fluorescence PCR detection is carried out according to the target fragment of the disease to be detected, the relative quantification is carried out by adopting a standard curve, the sample quality is judged, and accurate feasibility analysis and guidance are provided for whether the trace FFPE RNA sample can be used for subsequent RNA sequencing research. The trace FFPE RNA sample evaluation method breaks the application limit of the current main flow detection tool 2100Bioanalyzer, the lower limit of the total detectable sample amount can reach 50 pg/mu L, and the repeatability of the detection result is higher. Compared with the traditional 2100Bioanalyzer and other detection methods, the method provided by the application can directly display the abundance and availability of the target fragment, and is particularly suitable for transcriptome sequencing experiments of clinical FFPE samples. In addition, the method of the application does not need to additionally purchase expensive capillary electrophoresis analysis equipment, has lower cost and is easier to popularize.
Drawings
FIG. 1 is a graph showing the results of a 2100Bioanalyzer test in a comparative example of the present application;
FIG. 2 is a standard curve of the gene GUSB in the example of the present application;
FIG. 3 is a standard curve of the gene CDKN1A in the examples of the present application.
Detailed Description
The quality of a nucleic acid sample is measured, typically by measuring the amount of total nucleic acid in the sample to be measured, as well as its integrity and purity. However, in the detection of a gene against a certain disease, the amount and integrity of the gene of the disease to be detected are more important. Therefore, the application creatively proposes that for trace FFPE RNA samples, specific primers and probes of key genes of diseases to be detected are designed, and quality detection is carried out on the key genes through relative quantification of real-time fluorescence PCR, so that subsequent experiments and researches are convenient.
The real-time fluorescence PCR can relatively quantify the object to be detected, has high detection sensitivity and strong specificity, can rapidly and accurately evaluate the quality of some precious trace samples, has the detection lower limit of 50 pg/mu L, provides a rapid, low-cost and high-flux quality evaluation method for the quality detection of FFPE RNA samples, and also provides a more accurate reference basis for the subsequent transcriptome sequencing experimental analysis.
In the embodiment of the application, for quality detection of trace FFPE RNA samples of prostate cancer, it is understood that the quality detection method of trace FFPE RNA samples of the application is not limited to the trace FFPE RNA samples of prostate cancer, and trace FFPE RNA samples of other cancers are also suitable for the method of the application. In addition, the quality detection method of the trace RNA sample is not limited to the trace FFPE RNA sample and other trace samples to be detected, and the quality detection method can be used for detecting the quality of the trace FFPE RNA sample, and is not particularly limited.
The application is further illustrated by the following examples. The following examples are merely illustrative of the present application and should not be construed as limiting the application.
Examples
In this example, a trace FFPE RNA sample of prostate cancer is taken as an example for sample quality detection, and a sample to be tested of prostate cancer includes 24 trace FFPE RNA samples of FFPE sections of precious prostate cancer tissue, and a UHRR standard with the product number 740000 and the abbreviation of UHRR Universal Human Reference RNA purchased from Agilent Technologies is used as a standard. The details are as follows:
1. design of primers and probes
The two genes which have strong relativity with the occurrence of the prostate cancer and are often used as the key genes of the two genes which are the GUSB gene and the CDKN1A gene and are the main purposes of the transcriptome sequencing research of the prostate cancer are selected in the embodiment. Multiplex real-time fluorescent PCR primers and probes were designed for these two genes, the sequences of which are shown in Table 1.
TABLE 1 primers and probes for GUSB gene and CDKN1A gene
| Name of the name | Sequence (5 '. Fwdarw.3') | Seq ID No. |
| GUSB upstream primer | TGATCGCTCACACCAAATCC | 1 |
| GUSB downstream primer | CCTTGTCTGCTGCATAGTTAGAGTTG | 2 |
| GUSB probe | FAM-CTCCCGGCCTGTGACCTTTGTGA-TAMRA | 3 |
| CDKN1A upstream primer | CAAACACCTTCCAGCTCCTGTAA | 4 |
| CDKN1A downstream primer | AACGGGAACCAGGACACATG | 5 |
| CDKN1A probe | VIC-ATACTGGCCTGGACTGTTTTCTCTCGGC-TAMRA | 6 |
In both probes of Table 1, FAM and VIC are fluorescent groups, respectively, and TAMRA is a fluorescence quenching group.
2. Standard curve and real-time fluorescent PCR detection
Primers and probes of the GUSB gene and CDKN1A gene were mixed to prepare a 20 XPCR Assay, wherein the final concentration of each upstream primer was 600nM, the final concentration of each downstream primer was 600nM, and the final concentration of each probe was 300nM.
Real-time fluorescence PCR was performed using Taqman Fast Virus-step Master Mix from Thermo Scientific, with the following reaction system: taqman Fast Virus 1-step MasterMix 8.75. Mu.L, 20 XPCR Assay 1.75. Mu.L, nucleic acid sample 3.5. Mu.L, NF H 2 O21. Mu.L, after mixing, transfer to PCR plate, 10. Mu.L per well, 3 replicates per sample were set. The 96-well PCR plate spotting protocol is shown in table 2.
Table 2 96 well PCR plate spotting protocol
The reaction conditions are 50 ℃ for 20min and 95 ℃ for 2min, and 40 cycles are carried out: 95 ℃ for 5s and 60 ℃ for 60s. Fluorescence was collected at 60℃and fluorescence was collected from both FAM and VIC fluorescence channels, respectively.
3. Results and analysis
The results showed that the standard curve slope of gene GUSB = -3.298, r square = 0.999, amplification efficiency = 101.013, as shown in fig. 2. Standard curve slope of gene CDKN1A = -3.327, r square = 0.999, amplification efficiency = 99.776%, as shown in fig. 3. The relative concentrations of 24 samples to be tested obtained by converting the Ct values are shown in table 3.
TABLE 3 relative concentration of samples to be tested
In Table 3, samples 7, 14, 17, 18, 19 and 23 could not be used for downstream RNA sequencing experiments because the genes to be tested could not be detected or the concentration was below the lower limit of the detection range.
Comparative example
The same sample to be tested was used in this example for 2100Bioanalyzer assay, the detailed procedure was as follows:
(1) Taking 1 mu L of a sample to be detected, incubating for 2min at 70 ℃ on a PCR instrument, and immediately placing the sample on ice;
(2) Taking out the chip, adding 9.0 mu L of Gel-dye mix into the Gel hole bottom and injecting the chip by using an injection piston;
(3) 5.0. Mu.L of marker was added to each of the remaining 13 wells on the chip, and 1. Mu.L of spacer was added to the bottom of the spacer well;
(4) Adding 1 mu L of sample to be detected to the bottom of the sample hole, and vibrating and uniformly mixing the chip;
(5) Placing the chip into a 2100 biological analyzer, selecting a corresponding detection program, and running the program; experimental results were derived as shown in fig. 1 and table 4.
TABLE 4 detection results of samples to be tested using 2100Bioanalyzer
| Sequence number | Sample name | Concentration ng/. Mu.L | RIN | Sequence number | Sample name | Concentration ng/. Mu.L | RIN |
| 1 | 4964443 | 3.89 | 2.2 | 13 | 5266686 | 1.444 | 1 |
| 2 | 5260030 | 1.89 | 1.8 | 14 | 5266783 | 1.111 | 3.4 |
| 3 | 5260173 | 1.61 | 2 | 15 | 5266886 | 0.556 | 2.5 |
| 4 | 5262886 | 0.67 | 1 | 16 | 5266930 | 2.889 | 1.3 |
| 5 | 5263033 | 1.00 | 2.1 | 17 | 5267060 | 2.778 | 1 |
| 6 | 5263040 | 1.67 | 1.1 | 18 | 5267070 | 2.627 | 1.1 |
| 7 | 5263073 | 0.45 | 2.2 | 19 | 5267110 | 1.222 | 1 |
| 8 | 5263116 | 1.11 | 1 | 20 | 5293990 | 2.000 | 1.9 |
| 9 | 5263130 | 1.22 | 2.8 | 21 | 5294050 | 1.914 | 2.1 |
| 10 | 5263140 | 1.33 | 1.1 | 22 | 5294110 | 1.778 | 1 |
| 11 | 5266650 | 1.00 | 3.9 | 23 | 5294120 | 3.667 | 2.3 |
| 12 | 5266673 | 2.11 | 2.2 | 24 | 5294130 | 0.631 | 2.3 |
The results in FIG. 1 show that the gel trace is narrow and that 18S and 28S RNA are not visible. Since the sample concentration is lower than the detection threshold, the quantitative result is not credible; this also shows that the evaluation method of the present application is more sensitive and can evaluate lower concentration samples. In Table 4, RIN is much lower than 8 and is merely a description of total nucleic acid and cannot be used to guide the development of downstream NGS experiments. Taking sample 23 as an example, the 2100 concentration was quantitatively higher, but when the method of the present application was applied to the evaluation, GUSB was not detected and the relative concentration of CDKN1A was lower, so even if downstream NGS experiments were performed, the probability of detecting cancer-related information represented by GUSB and CDKN1A was extremely low.
This is because, although the trace FFPE RNA sample itself is highly fragmented, the 2100bioanalyzer results show that the concentration of FFPE RNA sample No. 23 is high enough for subsequent RNA sequencing, but 2100bioanalyzer cannot react to the degree of fragmentation, and according to the 2100bioanalyzer results, subsequent RNA sequencing is performed to detect prostate cancer related information represented by GUSB and CDKN1A, the detection rate is extremely low, and may not be detected at all. In the trace FFPE RNA sample evaluation method, if GUSB or CDKN1A is highly fragmented, a fluorescent signal cannot be detected, and if a relatively high quantitative signal is obtained, the GUSB and CDKN1A genes are proved to have certain integrity, and the method can be used for subsequent RNA sequencing detection. Therefore, the evaluation method can provide and display more accurate sample quality evaluation, and is more suitable for quality evaluation of trace FFPE RNA samples for RNA sequencing.
The foregoing is a further detailed description of the application in connection with specific embodiments, and it is not intended that the application be limited to such description. It will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the application, and these should be considered to be within the scope of the application.
SEQUENCE LISTING
<110> Shenzhen Hua institute of great life science
<120> method for evaluating trace FFPE RNA sample, kit and application
<130> 17I24583
<160> 6
<170> PatentIn version 3.3
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<211> 20
<212> DNA
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Claims (3)
1. A method for trace FFPE RNA sample evaluation of prostate cancer, characterized in that: selecting a key gene of the prostate cancer, and adopting a specific primer and a probe of the key gene to perform real-time fluorescence PCR detection on a trace FFPE RNA sample to be detected;
analyzing the real-time fluorescence PCR detection result by adopting a standard curve to obtain the relative concentration and relative abundance of the genes related to the prostate cancer in the trace FFPE RNA sample, thereby evaluating the usability of the trace FFPE RNA sample;
the key genes of the prostate cancer comprise a GUSB gene and a CDKN1A gene, and the real-time fluorescence PCR detection is multiplex real-time fluorescence PCR of the two genes;
the upstream primer and the downstream primer of the specific primer of the GUSB gene are respectively a sequence shown by a Seq ID No.1 and a sequence shown by a Seq ID No.2, and the specific probe of the GUSB gene is a sequence shown by a Seq ID No. 3; the upstream primer and the downstream primer of the specific primer of the CDKN1A gene are respectively a sequence shown by a Seq ID No.4 and a sequence shown by a Seq ID No.5, and the specific probe of the CDKN1A gene is a sequence shown by a Seq ID No. 6;
Seq ID No.1：5’- TGATCGCTCACACCAAATCC -3’
Seq ID No.2：5’- CCTTGTCTGCTGCATAGTTAGAGTTG -3’
Seq ID No.3：5’- FAM-CTCCCGGCCTGTGACCTTTGTGA-TAMRA -3’
Seq ID No.4：5’- CAAACACCTTCCAGCTCCTGTAA -3’
Seq ID No.5：5’- AACGGGAACCAGGACACATG -3’
Seq ID No.6：5’- VIC-ATACTGGCCTGGACTGTTTTCTCTCGGC-TAMRA -3’。
2. the method according to claim 1, characterized in that: the standard curve is obtained by amplifying an RNA standard substance by adopting the same real-time fluorescence PCR as the trace FFPE RNA sample.
3. The method according to claim 2, characterized in that: the RNA standard is UHRR human standard.
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