4 results about "Reverse Transcription Process" patented technology

The use of different primer sets in reverse transcription incorporates tags allowing for the selective amplification of cDNA transcribed using the different primers. Primers targeting non-desired RNA sequences such as ribosomal RNA can be used to prevent subsequent amplification of cDNA transcribed from those non-coding fragments. Accordingly effective depletion of non-desired sequences after cDNA amplification can be achieved. Systems and methods of the invention have applications in whole-transcriptome analysis. Non-coding sequence targeting primers can include nucleotide analogs that, when enzymatically processed, prevent subsequent amplification. Library preparation can include single primer isothermal amplification (SPIA) techniques wherein an RNA sequence required for SPIA is included in random primers but is absent from primers targeting non-coding RNA.

The present application provides a kit and method for detecting RNA at room temperature. The kit for detecting RNA at room temperature comprises: a target RNA recognition and amplification component and a double signal amplification component, and the further amplification and reporting of the signal are completed by coupling the two components, and the high-sensitivity detection of RNA is realized. The method for detecting RNA at room temperature of the present application does not need a complex reverse transcription process, can reduce the complexity of the reaction and the cost of the instrument, and has high detection efficiency.

Disclosed herein are methods for performing in situ sequencing of RNA transcripts with non-uniform 5′ ends. During reverse transcription (RT) of RNA transcripts, RT enzyme is induced to “template-switch” to a separate oligonucleotide provided as the template for the upstream flanking region. This flanking region is grafted onto the beginning of the cDNA, enabling padlock probe detection, rolling circle amplification, and fluorescent in situ sequencing. Overall, the disclosed method for in situ sequencing can be applicable for analyzing exogenously introduced transcripts (e.g., identifying and determining impact of a perturbation including a CRISPR perturbation or shRNA / siRNA / ASO perturbation), analyzing naturally occurring transcripts (e.g., measuring gene expression, detecting splicing events), and analyzing modified, naturally occurring transcripts (e.g., detecting mutations or gene edits).

The invention discloses a heat-resistant MLV reverse transcriptase mutant as well as a preparation method and application thereof. The invention discloses an MLV reverse transcriptase mutant with an amino acid sequence as shown in SEQ ID NO.1 at first. The invention further discloses a preparation method and application of the MLV reverse transcriptase mutant. According to the MLV reverse transcriptase mutant, key amino acid sites of wild type MLV reverse transcriptase are modified through site-specific mutagenesis, the thermal stability of the MLV reverse transcriptase in the range of 45-65 DEG C is improved, the optimum temperature in the reverse transcription process is increased to 65 DEG C from 45 DEG C, meanwhile, the low RNase H activity of the MLV reverse transcriptase mutant is kept, degradation of RNA-cDNA hybrid chains is reduced, and the MLV reverse transcriptase mutant has the advantages of being high in yield and high in stability. The technical effects of improving the reverse transcription reaction efficiency, enhancing the reverse transcription capability on a complex secondary structure RNA template and reducing the non-specific reaction are achieved.