scholarly journals Non-biased and efficient global amplification of a single-cell cDNA library

2013 ◽  
Vol 42 (2) ◽  
pp. e12-e12 ◽  
Author(s):  
Huan Huang ◽  
Mari Goto ◽  
Hiroyuki Tsunoda ◽  
Lizhou Sun ◽  
Kiyomi Taniguchi ◽  
...  
Keyword(s):  
Single Cell ◽  
Cdna Library ◽  
Global Amplification

scholarly journals Global cDNA Amplification Combined with Real-Time RT–PCR: Accurate Quantification of Multiple Human Potassium Channel Genes at the Single Cell Level

Yeast ◽  
2000 ◽  
Vol 1 (3) ◽  
pp. 201-210 ◽  
Author(s):  
A. Al-Taher ◽  
A. Bashein ◽  
T. Nolan ◽  
M. Hollingsworth ◽  
Brady G.
Keyword(s):  
Potassium Channel ◽  
Single Cell ◽  
Real Time ◽  
Quantitative Pcr ◽  
Single Cells ◽  
Initial Step ◽  
Small Samples ◽  
Rt Pcr ◽  
Real Time Quantitative Pcr ◽  
Global Amplification

We have developed a sensitive quantitative RT–PCR procedure suitable for the analysis of small samples, including single cells, and have used it to measure levels of potassium channel mRNAs in a panel of human tissues and small numbers of cells grown in culture. The method involves an initial global amplification of cDNA derived from all added polyadenylated mRNA followed by quantitative RT–PCR of individual genes using specific primers. In order to facilitate rapid and accurate processing of samples, we have adapted the approach to allow use ofTaqMan™real-time quantitative PCR. We demonstrate that the approach represents a major improvement over existing conventional and real-time quantitative PCR approaches, since it can be applied to samples equivalent to a single cell, is able to accurately measure expression levels equivalent to less than 1/100th copy/cell (one specific cDNA molecule present amongst108total cDNA molecules). Furthermore, since the initial step involves a global amplification of all expressed genes, a permanent cDNA archive is generated from each sample, which can be regenerated indefinitely for further expression analysis.

cDNA library preparation from total RNA extracts of Single-cell marine protists (e.g. Acantharia, Strombidium basimorphum, and Prymnesium parvum) for transcriptome sequencing v2

2022 ◽  
Author(s):  
Joost S S. Mansour ◽  
Konstantinos Anestis ◽  
Fabrice Not ◽  
Uwe John
Keyword(s):  
Single Cell ◽  
Cdna Library ◽  
Mammalian Cells ◽  
Single Cells ◽  
Rna Extraction ◽  
Cdna Libraries ◽  
Prymnesium Parvum ◽  
Large Variability ◽  
Total Rna ◽  
Marine Protists

Many marine protists are not culturable and therefore challenging to study, nonetheless, they are essential in all marine ecosystems. The development of single-cell techniques is allowing for more marine protists to be studied. Such genomic approaches aim to help to disentangle heterotrophic processes such as phagotrophy from osmotrophy and phototrophic-induced anabolic activities. This information will then support cellular and metabolic modeling by better elucidating the physiological mechanisms and quantifying their importance in different scenarios. However, single-cell protocols and low input RNA kits for transcriptomics are usually made for and tested with mammalian cells, as such the feasibility and efficiency of single-cell transcriptomics on highly diverse mixotrophic protists is not always known. Often single-cell transcriptomics of microbial eukaryotes shows low transcript recovery rates and large variability. We report on transcriptomic methods that we have successfully performed on single cells of Acantharia, Strombidium basimorphum, and Prymnesium parvum. This protocol follows up after total RNA extraction (from the protocol at dx.doi.org/10.17504/protocols.io.bp6xmrfn) to prepare cDNA libraries for Illumina sequencing. The described protocol uses the SMART-Seq4 kit (Takara #634891) for cDNA synthesis and amplification, but this can also be successfully performed with the NEBNext kit (NEB #E6421). The NEBNext kit protocol is very similar to the protocol described here and generally the manufacture's protocol can be followed but see the notes at step 4 and step 18 of this protocol, and do the final elution after cDNA purification in 10 mM Tris (pH 8.0). The subsequent cDNA library is prepared following the .

scholarly journals Chum-RNA allows preparation of a high-quality cDNA library from a single-cell quantity of mRNA without PCR amplification

2008 ◽  
Vol 36 (15) ◽  
pp. e92-e92 ◽  
Author(s):  
Takahiro Tougan ◽  
Daisuke Okuzaki ◽  
Hiroshi Nojima
Keyword(s):  
Single Cell ◽  
Cdna Library ◽  
Pcr Amplification ◽  
High Quality

scholarly journals Global cDNA Amplification Combined with Real-Time RT–PCR: Accurate Quantification of Multiple Human Potassium Channel Genes at the Single Cell Level

Yeast ◽  
2000 ◽  
Vol 1 (3) ◽  
pp. 201-210 ◽  
Author(s):  
A. Al-Taher ◽  
A. Bashein ◽  
T. Nolan ◽  
M. Hollingsworth ◽  
Brady G.
Keyword(s):  
Potassium Channel ◽  
Single Cell ◽  
Real Time ◽  
Quantitative Pcr ◽  
Single Cells ◽  
Initial Step ◽  
Small Samples ◽  
Rt Pcr ◽  
Real Time Quantitative Pcr ◽  
Global Amplification

We have developed a sensitive quantitative RT–PCR procedure suitable for the analysis of small samples, including single cells, and have used it to measure levels of potassium channel mRNAs in a panel of human tissues and small numbers of cells grown in culture. The method involves an initial global amplification of cDNA derived from all added polyadenylated mRNA followed by quantitative RT–PCR of individual genes using specific primers. In order to facilitate rapid and accurate processing of samples, we have adapted the approach to allow use of TaqMan™ real-time quantitative PCR. We demonstrate that the approach represents a major improvement over existing conventional and real-time quantitative PCR approaches, since it can be applied to samples equivalent to a single cell, is able to accurately measure expression levels equivalent to less than 1/100th copy/cell (one specific cDNA molecule present amongst 108 total cDNA molecules). Furthermore, since the initial step involves a global amplification of all expressed genes, a permanent cDNA archive is generated from each sample, which can be regenerated indefinitely for further expression analysis.

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