cdna synthesis
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2021 ◽  
Author(s):  
Ahmed Ibrahim

Abstract To inhibit HIV replication and infection, we have designed novel linear single stranded modified antisense nucleic acid oligonucleotides ending with or without chain terminating bases (Which resemble the shape of the comb). They were targeting specifically the HIV-1 clone pNL4-3 strong promoter pre PBS region to stop cDNA synthesis within or before the R region, preventing the viral reverse transcriptase (RT) jumping to the 3' end and continue copying the virus. The main advantages of our comb shaped oligonucleotides are their specificity and extreme protection against resistance by known viral mutations. Promising results were obtained for two 15-mer compounds at one tenth azidothymidine concentration. As a result we claim that when adapted properly, the comb shaped antivirals can be used to target the genomic RNA of a number of serious viruses such as for example Ebola, SARS-CoV-2, Influenza, Dengue, hepatitis C, Chikungunya and Zika as they are all using polymerases to copy their genomic RNA1-8. Their genomic RNA could be destroyed through the human or viral endonucleases instead of the viral RT RNAseH site when their polymerases are stopped at specific sites.


2021 ◽  
Vol 5 (2) ◽  
pp. 24
Author(s):  
Dino Pećar ◽  
Ivana Čeko ◽  
Lana Salihefendić ◽  
Rijad Konjhodžić

Monitoring of the lineages SARS-CoV-2 is equally important in a fight against COVID-19 epidemics, as is regular RT - PCR testing. Ion AmpliSeq Library kit plus is a robust and validated protocol for library preparation, but certain optimizations for better sequencing results were required. Clinical SARS-CoV-2 samples were transported in three different viral transport mediums (VTM), on arrival at the testing lab, samples were stored on -20OC. Viral RNA isolation was done on an automatic extractor using a magnetic beads-based protocol. Screening for positive SARS-CoV-2 samples was performed on RT–PCR with IVD certified detection kit. This study aims to present results as follows: impact of first PCR cycle variation on library quantity, comparison of VTMs with a quantified library, maximum storage time of virus and correlation between used cDNA synthesis kit with generated target base coverage. Our results confirmed the adequacy of the three tested VTMs for SARS-CoV-2 whole-genome sequencing. Tested cDNA synthesis kits are valid for NGS library preparation and all kits give good quality cDNA uniformed in viral sequence coverage. Results of this report are useful for applicative scientists who work on SARS-CoV-2 whole-genome sequencing to compare and apply good laboratory practice for optimal preparation of the NGS library.


2021 ◽  
Author(s):  
Padmini Ramachandran ◽  
Tamara Walsky ◽  
Amanda Windsor ◽  
maria.hoffmann not provided ◽  
Chris Grim

PURPOSE: This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR, library prep, genome sequencing, quality control checks, and data submission to NCBI. This modified protocol details methods for cDNA synthesis and library preparation for sequencing of wastewater samples containing SARS-CoV-2. The protocol is based primarily on the NEBNext® ARTIC SARS-CoV-2 Library Prep Kit (Illumina®), NEB #E7650S/L 24/96 reactions, with a few modifications. Primarily, VarSkip Short primers are used in place of the ARTIC V3 primers. These primers are available in the NEBNext®ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina®); however, for optimal variant detection from wastewater, sequenced fragments should be as large as possible, so we discourage fragmentation prior to end prep. There are a couple of decision points in this protocol. Examining cDNA amplicon samples on an Agilent TapeStation system or similar fragment analyzer is extremely helpful in making these decisions.


2021 ◽  
Author(s):  
Ajeet Kumar ◽  
Vatsal Mishra ◽  
Chandra Bhan Singh ◽  
Rashmi Patel ◽  
Siddharth Samrat ◽  
...  

Abstract Background Chronic myeloid leukaemia (CML) is a hematopoietic stem cell disorder, caused by a balanced reciprocal translocation (t(9;22) (q34;q11))that lead to the formation of BCR (Break point Cluster Region)-ABL (Abelson) fusion transcripts known as Philadelphia (Ph) chromosome. Prevalence of BCR-ABL fusion transcripts in Indian CML population is poorly understood and few studies have been reported from India. The aim of present study was to determine the frequencies as well as prognostic effects of the three fusion transcripts i.e. b2a2, b3a2 and e1a2 in an Indian population. Methods RNA was isolated from total 123 sample 27 bone marrow (BM) sample and 96 Peripheral blood (PB) sample of CML patient followed by cDNA synthesis. Real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed using TaqMan→ assay (ABI, CA, USA) to monitor BCR-ABL transcript. Results Ph' chromosome was observed in 103 patients whereas it was not detected in 20 cases. qRT-PCR revealed that the b3a2 fusion transcripts was the most common transcript in CML patients (63.41%) while b2a2 fusion transcript was present in 16.26% cases. Co-expression of b3a2+b2a2 fusion transcript was observed in 0.81% cases whereas co-expression of b3a2+e1a2 fusion transcript was found in 1.63% cases. There was no co-relation observed between b3a2 fusion transcript and platelet count. The fusion transcript b2a2 was observed in relatively younger patients compared to b3a2 fusion transcript. Although this correlation was not statistically significant. Conclusion The co-expression of BCR-ABL fusion transcripts was higher (63.41% aggregate of b3a2) in the present population in contrast to other populations reported. This finding was consistent with the frequency data reported from Sudan.


2021 ◽  
Author(s):  
Michael Eadon ◽  
Ricardo Melo Ferreira ◽  
Ying-Hua Cheng ◽  
Tarek Ashkar
Keyword(s):  

The tissue in OCT undergoes cryosectioning, affixment to the cDNA capture slide, H+E staining, 20x Keyence imaging, tissue permeabilization, RNA capture, and cDNA synthesis. Data is analyzed in Loupe browser and in R prior to uploading to the KPMP Data Lake.


2021 ◽  
Author(s):  
Michael Eadon ◽  
Ricardo Melo Ferreira ◽  
Ying-Hua Cheng ◽  
Tarek Ashkar
Keyword(s):  

The tissue in OCT undergoes cryosectioning, affixment to the cDNA capture slide, H+E staining, 20x Keyence imaging, tissue permeabilization, RNA capture, and cDNA synthesis. Data is analyzed in Loupe browser and in R prior to uploading to the KPMP Data Lake.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shreya Ghimire ◽  
Carley G. Stewart ◽  
Andrew L. Thurman ◽  
Alejandro A. Pezzulo

AbstractRNA sequencing enables high-content/high-complexity measurements in small molecule screens. Whereas the costs of DNA sequencing and RNA-seq library preparation have decreased consistently, RNA extraction remains a significant bottleneck to scalability. We evaluate the performance of a bulk RNA-seq library prep protocol optimized for analysis of many samples of adherent cultured cells in parallel. We combined a low-cost direct lysis buffer compatible with cDNA synthesis (in-lysate cDNA synthesis) with Smart-3SEQ and examine the effects of calmidazolium and fludrocortisone-induced perturbation of primary human dermal fibroblasts. We compared this method to normalized purified RNA inputs from matching samples followed by Smart-3SEQ or Illumina TruSeq library prep. Our results show the minimal effect of RNA loading normalization on data quality, measurement of gene expression patterns, and generation of differentially expressed gene lists. We found that in-lysate cDNA synthesis combined with Smart-3SEQ RNA-seq library prep generated high-quality data with similar ranked DEG lists when compared to library prep with extracted RNA or with Illumina TruSeq. Our data show that small molecule screens or experiments based on many perturbations quantified with RNA-seq are feasible at low reagent and time costs.


Author(s):  
Zahra Sokhanvar ◽  
Ameneh Elikaei ◽  
Zohreh Sharifi

Background: Liver disease is more severe in HDV+HBV co-infected patients than HBV infected patients which seems to be related to differences in the expression of genes and other factors such as MicroRNAs (miRNAs). The aim of this study was to investigate miR-222 expression in HBV infected patients in comparison with HDV+HBV co-infected patients. Methods: First, total RNA was extracted from the serum samples and then, complementary DNA (cDNA) was produced using cDNA synthesis kit. Finally, miR-222 gene expression was measured using U6 as the internal control by quantitative PCR (qPCR). Results: The level of miR-222 expression in HDV+HBV co-infected samples was significantly up regulated. The fold change of the miR-222 expression between two groups was 3.3 (95% CI; 0.011- 17.63) with p<0.001. Conclusion: The expression of miR-222 was higher in HBV+HDV co-infected patients than HBV infected patients. Further studies should be conducted to confirm whether miR-222 can be a biomarker for prognosis of severe liver diseases.


2021 ◽  
Author(s):  
New England not provided Biolabs

This protocol details methods for the NEBNext® ARTIC SARS-CoV-2 RT-PCR Module, NEB #E7626S/L 24/96 reactions. cDNA Synthesis and Targeted cDNA Amplification with NEBNext VarSkip Short Primer Mixes: This protocol follows an alternate variant-tolerant approach for targeting SARS-CoV-2 by utilizing NEBNext VarSkip Short SARS-CoV-2 Primer Mixes. The NEBNext VarSkip Short SARS-CoV-2 Primer mixes cannot be added to the same cDNA amplification reaction as the NEBNext ARTIC SARS-CoV-2 Primer Mixes. If downstream applications include sequencing, performing RNA input normalization prior to cDNA synthesis and targeted amplification promotes more even distribution of reads across sequencing libraries. For other NEBNext® ARTIC SARS-CoV-2 protocols, please see the NEBNext ARTIC Protocols Collection. To obtain instructions for using NEBNext ARTIC SARS-CoV-2 Primer Mix and the NEBNext® ARTIC SARS-CoV-2 RT-PCR Module please see the NEBNext ARTIC SARS CoV2 RT PCR Module Manual.


2021 ◽  
Author(s):  
New England not provided Biolabs

This protocol details methods for the NEBNext® ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina®), NEB #E7658S/L 24/96 reactions. This protocol follows an alternate variant-tolerant approach for targeting SARS-CoV-2 by utilizing NEBNext VarSkip Short SARS-CoV-2 Primer Mixes. The NEBNext VarSkip Short SARS-CoV-2 Primer mixes cannot be added to the same cDNA amplification reaction as the NEBNext ARTIC SARS-CoV-2 Primer Mixes. This protocol does not include a cleanup step for each sample after cDNA synthesis and after adaptor ligation. Performing RNA input normalization prior to cDNA synthesis and targeted amplification and/or normalizing final libraries prior to sequencing promotes more even distribution of reads across libraries. Skipping RNA input normalization, final library normalization, and cleanups reduces hands on time but may require deeper sequencing depth to reach sufficient coverage of each sample. For other NEBNext® ARTIC SARS-CoV-2 protocols, please see the NEBNext ARTIC Protocols Collection.


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