An efficient and sensitive method for preparing cDNA libraries from scarce biological samples
نویسندگان
چکیده
The preparation and high-throughput sequencing of cDNA libraries from samples of small RNA is a powerful tool to quantify known small RNAs (such as microRNAs) and to discover novel RNA species. Interest in identifying the small RNA repertoire present in tissues and in biofluids has grown substantially with the findings that small RNAs can serve as indicators of biological conditions and disease states. Here we describe a novel and straightforward method to clone cDNA libraries from small quantities of input RNA. This method permits the generation of cDNA libraries from sub-picogram quantities of RNA robustly, efficiently and reproducibly. We demonstrate that the method provides a significant improvement in sensitivity compared to previous cloning methods while maintaining reproducible identification of diverse small RNA species. This method should have widespread applications in a variety of contexts, including biomarker discovery from scarce samples of human tissue or body fluids.
منابع مشابه
Improved Procedure for Screening Expression Libraries for Novel Autoantigens
The standard method for immunoscreening of a cDNA expression library is time-consuming becauseof the production of a large proportion of false positives during the first and second round of screening.This problem is more important when a sensitive chemiluminescence detection system is used. Due tothe high sensitivity of the detection system, there is a need to avoid false posi...
متن کاملRepresentative in Vitro cDNA Amplification From Individual Hemopoietic Cells and Colonies
A simple procedure is described for preparing microgram amounfs of cDNA, suitable for cloning into libraries, from samples as small as a single cell. Synthesis of cDNA is primed with oligo(dT) and the resulting strands are tailed with poly(dA). The cDNA is then amplified using an oligo(dT)-containing sequence to prime the polymerase chain reaction. By limiting the size of the first cDNA strands...
متن کاملDevelopment of a Simple and Efficient Method for Preconcentration and Determination of Trace Levels of Fexofenadine in Plasma and Urine Samples
A simple, inexpensive and sensitive three-phase hollow fiber liquid-phase microextraction (HFLPME) coupled with high performance liquid chromatography (HPLC) with UV detection was successfully developed for determination of trace level of an anti-Histamine drug, fexofenadine, in human plasma and urine. The analyte was extracted into n-octanol that was immobilized in the wall of a porous hollow ...
متن کاملDEVELOPMENT OF A RAPID AND SENSITIVE RADIOIMMUNOASSAY FOR MEASUREMENT OF AFLATOXIN B 1 IN BIOLOGICAL SAMPLES
Aflatoxin B I (AFB) isa well known hepatocarcinogen in several animal species and probably a causative agent in human hepatocellular carcinoma. Humans are exposed to AFB by ingesting contaminated food. Aflatoxin contamination encountered in human foods is usually at low levels which is difficult to measure by chromatographic methods. Therefore in the present study we have developed an immun...
متن کاملConstruction and Evaluation of Normalized cDNA Libraries Enriched with Full-Length Sequences for Rapid Discovery of New Genes from Sisal (Agave sisalana Perr.) Different Developmental Stages
To provide a resource of sisal-specific expressed sequence data and facilitate this powerful approach in new gene research, the preparation of normalized cDNA libraries enriched with full-length sequences is necessary. Four libraries were produced with RNA pooled from Agave sisalana multiple tissues to increase efficiency of normalization and maximize the number of independent genes by SMART&tr...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
عنوان ژورنال:
دوره 43 شماره
صفحات -
تاریخ انتشار 2015