The transcriptome is a collection of RNA molecules that reveals the transcription products of all genes in an organism, i.e., all RNA molecules including transcribed RNAs and non-coding RNAs. Through transcriptome data, we can study the dynamic expression of genes and the regulation of gene expression at a certain period or a certain state of the organism from the RNA level, and the transcriptome data can be combined with genomic and proteomic data to analyze the biological transcripts in a more comprehensive way, which can help us to understand the degree of activity and function of the genes in different biological processes, environmental conditions, or disease states.
Currently, Transcriptome Analysis Methods include a variety of techniques, but each technique has its advantages and limitations, some common methods are as follows:
RNA-array
RNA-array is hybridization-based sequencing. The principle is to fix the known gene templates in a certain order or arrangement on a solid phase carrier such as membrane, slide or silicon, then extract the RNA of the control and experimental samples and reverse transcribe them into cDNA, after reverse transcription, use different fluorescent dyes to label the cDNAs of the control and experimental groups (the excitation light of the control group is green, and the excitation light of the experimental group is red), and then hybridize the cDNAs produced by reverse transcription with the fixed gene templates one by one to produce base complementary pairing, and finally elute the unbound cDNAs by hybridization. Then the cDNA from reverse transcription will be hybridized with the pre-fixed gene template one by one to generate base complementary pairing, and the unbound cDNA will be eluted, and finally the light signals at each site under the laser will be detected by laser confocal microscope and other equipments, and the different colors of the light signals will be used to determine the difference in the expression of a certain gene between the two groups of samples. For example, if the ratio of gene expression of gene A in the control group to that of the experimental group is 1:1, the green light and red light will appear yellow when taking pictures; if the ratio of gene expression of gene B in the control group to that of the experimental group is greater than 1 (e.g., 30:1), the red light will appear a slightly darker red when taking pictures with the red light and green light overlapping. In this way we can detect the expression levels of different genes and identify differentially expressed genes.
The steps of the RNA-array experiment are as follows:
(1) Firstly, extract the total RNA from the control group and the cells or tissues to be tested; (2) Use reverse transcriptase to reverse transcribe the RNA into cDNA, and add different fluorescent markers to label the cDNA of the control group and the experimental group; (3) Hybridize the labeled cDNA with the pre-designed microarray gene chip, on which there are a number of different probes that have already been fixed and each of them corresponds to a specific gene or transcript. Each probe corresponds to a specific gene or transcript; (4) Scanning the microarray using a scanner to detect the light signal at each site and thus obtain the fluorescence signal intensity of each probe, which can reflect the expression level of a specific gene or transcript in the sample to be detected as well as the level of differential expression; (5) Data analysis of the scanning results using a specialized software, including data preprocessing (e.g., normalization, background correction, etc.), quality assessment, and analysis of the results of the scanning, background correction, etc.), quality assessment and differential expression analysis.
RNA-array experiment has the following advantages and disadvantages: (1) Advantages: the operation is relatively simple, suitable for detecting the expression level of known genes; if the designed probe is longer, it can increase the specificity; more than 30,000 different genes can be detected at one time.
(2) Limitations: because of the need to design probes in advance, it is not possible to detect unknown genes and new transcripts, and the sensitivity is lower; it is not possible to detect transcripts with lower abundance; there are cross-hybridization and non-specific hybridization of the probes, and other phenomena.
RNA-Seq
RNA-Seq is the most advanced transcriptome analysis technology, which can directly sequence genomic cDNA by combining with high-throughput sequencing technology, calculate the expression of different RNAs by counting the number of related Reads (small cDNA fragments for sequencing), and generate the complete sequence information of transcripts.
The steps of RNA-Seq experiment are as follows: (1) extract the total RNA of the tissue or cell to be tested and test the purity, concentration and integrity of the RNA; (2) if you are testing the transcription of the mRNA, take advantage of the feature that eukaryotic mature mRNA contains 5' polyA tails to use oligo dT beads to specifically bind to the poly(A) tails of the mRNA. Specifically bind to the poly(A) tail of mRNA and isolate the mRNA from the total RNA, and then purify and fragment the isolated RNA; (3) Use reverse transcriptase to reverse transcribe the RNA into cDNA and purify the double-stranded cDNA; (4) Construct the cDNA library, and add a different junction to each sample, i.e., it possesses a different index, so that different samples can be differentiated from each other according to different indexes; (5) Construct a library of different samples; (6) Use oligo dT magnetic beads to bind to the poly(A) tail of mRNA. different sample libraries; (5) use primers with P5, P7 and barcodej for PCR to enrich libraries, purify libraries and carry out quality control; (6) analysis of biosignature tools, such as data quality control, reference genome comparison, expression and differentially expressed genes, and functional enrichment.
RNA-Seq experiment has the following advantages and disadvantages, (1) Advantages: able to comprehensively and accurately analyze the transcriptome, including known and unknown genes; can detect the transcripts of a species in a specific environment or a certain period of time; detect the variable splicing events of the transcripts and the information of the gene structure variations, gene fusion, etc.; can detect the low abundance of transcripts. (2) Limitations: Higher technical requirements and data analysis ability are required, and the cost is higher; most of the extracted transcripts have mitochondrial and ribosomal RNA, which restricts the reading and expression analysis of other RNAs; RNA isolation technology has errors.
Quantitative PCR
Quantitative PCR is a method of detecting the total amount of product after the PCR cycle by adding fluorescent chemicals to the DNA amplification reaction. It takes advantage of the fact that DNA polymerizes into new DNA strands during the PCR process, adds a fluorescent-labeled probe or dye to bind the newly synthesized DNA strands, and measures the PCR reaction by monitoring the intensity of the fluorescent signal in real time, and then quantitatively analyzes the specific DNA sequences in the samples to be tested by using the internal reference method or the external reference method.Detection of RNA expression levels of specific genes by real-time PCR is commonly used for genetic validation of small samples.
Advantages and disadvantages of Quantitative PCR, (1) Advantages: high sensitivity, able to accurately measure the expression level of specific genes; can y verify the reliability of RNA-seq and microarray results; low requirements for sample quality and quantity, simple and easy to operate. (2) Limitations: only a limited number of genes can be analyzed, whole transcriptome analysis is not possible; manually designed primers may have false positives.
Northern Blot
Northern Blot is a technique used to detect the expression level of target genes. The principle is to perform denaturing agarose gel electrophoresis on the RNA of the sample to be tested, distinguish RNA molecules according to different molecular weights, and then transfer the RNA molecules to the nitrocellulose membrane. And use a pre-designed probe to hybridize with it to detect the presence and number of specific RNA.
Northern Blot experimental procedure: (1) Extract the total RNA of the sample to be tested and purify; (2) RNA formaldehyde denaturation gel electrophoresis was used to separate RNA with different molecular weight. (3) RNA was transferred to the nitrocellulose membrane and fixed by baking or UV cross-linking; (4) The probe was hybridized with RNA and the expression level was detected.
Advantages and disadvantages of Northern Blot, (1) Advantages : strong specificity, suitable for verifying the size and expression of RNA; it can detect the expression of different genes in the same tissue cells or detect the expression level of the same gene between different tissue cells. (2) Limitations: complicated technical operation and low sensitivity; only a limited number of RNA can be detected.
Single-Cell RNA-Seq
Almost all cells of multicellular organisms have the same genetic material, but the transcriptome information of each cell varies due to different cell states or periods. The transcript of a single cell can truly reflect the cell identity and function. Single-cell RNA-Seq technology is to reveal the transcriptome characteristics of each cell in the cell population by sequencing the RNA in a single cell. The first step in the Single-Cell RNA-Seq experimental process is to isolate and capture high-quality single cells from tissues or cell populations for detection, and the remaining steps are similar to RNA-seq.
Advantages and disadvantages of Single-Cell RNA-Seq, (1) Advantages: it can capture cell heterogeneity, is suitable for cell subgroup analysis in complex tissues, and identify rare cell types; the flux can detect hundreds of thousands of cells. (2) Limitations: the technology is more complex, data analysis is difficult, the cost is higher.
RNA-Seq of single cells(Hrdlickova R et al.,2018)
Spatial transcriptome sequencing
The spatial distribution of gene expression within an organism's tissues or cells can be explored using spatial transcriptome sequencing technology, which allows RNA sequencing while preserving RNA spatial information. Currently, there are two main directions of spatial transcriptome technology: (1) the first is based on second-generation sequencing (NGS), the core of which is to tag short sequences with spatial information (spatial barcode/ID) to the sequenced reads from different locations, including the solid-phase transcriptome capture technology (with a large range of capture to obtain more information) and deterministic spatial barcodes (more flexible and less costly than the former), but it is more flexible and less costly than the former. (more flexible and lower cost than the former), but the common point is to collect spatial barcode RNA and sequencing; (2) the second is based on image (Image), the main way to complete the in situ amplification supplemented by in situ sequencing/in situ hybridization technology to complete the principle is that single RNA molecules in the natural position can emit fluorescent signals, with the help of high-resolution fluorescence microscope can detect and distinguish single molecules generated from different RNA molecules. With the help of high-resolution fluorescence microscope, we can detect and differentiate the single molecule signals generated from different RNA molecules, and then use image processing to generate the gene expression matrix.
Advantages and disadvantages of spatial transcriptome sequencing, (1) Advantages: it can detect the spatial information of gene expression, and reveal the location information and interactions of genes in different tissues or cells; (2) Disadvantages: its technology is more complicated, data processing is more difficult, and the cost is higher.
In conclusion, each of these methods has its own advantages and disadvantages, and in practice these techniques are often complementary and can be used synergistically, so the appropriate technique can be selected according to the objectives of the study and the budget.
References:
- Kharchenko PV. "The triumphs and limitations of computational methods for scRNA-seq. "Nat Methods. 2021;18(7):723-732.https://pubmed.ncbi.nlm.nih.gov/34155396/
- Moffitt JR, Lundberg E, Heyn H. "The emerging landscape of spatial profiling technologies." Nat Rev Genet. 2022;23(12):741-759.https://pubmed.ncbi.nlm.nih.gov/35859028/
- Hrdlickova R, Toloue M, Tian B. "RNA-Seq methods for transcriptome analysis." Wiley Interdiscip Rev RNA.201;8(1):10.1002/wrna.1364.https://pubmed.ncbi.nlm.nih.gov/27198714/
