isolation-of-precursor-b-cell-subsets-from-umbilical-cord-blood

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

University of Missouri-Columbia

Researchers whose experimental models are mammalian oocytes and preimplantation embryos are often limited by the yield of nucleic acids that can be isolated from such a small sample size. In addition, the limited number of cells from these types of samples makes the simultaneous recovery of RNA and DNA very difficult and often sample pooling is necessary to increase nucleic acid yield. Here we report a simple set of procedures using commercially available kits that results in consistent yield and quality of nucleic acids. After sample lysis, RNA is isolated and converted to a reusable cDNA library. Following RNA isolation, DNA is precipitated, isolated, and bisulfite converted for DNA methylation studies. Our results demonstrate the feasibility of isolating RNA and DNA from a small number of cells with repeatability of results.

Isolation of RNA and DNA from single preimplantation embryos and a small number of Mammalian oocytes for imprinting studies.

Chercheurs, dont des mod&egrave;les exp&eacute;rimentaux sont des mammif&egrave;res ovocytes et embryons pr&eacute;implantatoires sont souvent limit&eacute;s par le rendement d&#39;acides nucl&eacute;iques qui peuvent &ecirc;tre isol&eacute;s dans une telle taille de petit &eacute;chantillon. En outre, le nombre limit&eacute; de cellules provenant de ces types d&#39;&eacute;chantillons rend la reprise simultan&eacute;e de l&#39;ARN et l&#39;ADN tr&egrave;s difficile et souvent l&#39;&eacute;chantillon en commun est n&eacute;cessaire pour augmenter le rendement de l&#39;acide nucl&eacute;ique. Nous rapportons ici un simple ensemble de proc&eacute;dures utilisant des kits disponibles dans le commerce qui se traduit par une production coh&eacute;rente et la qualit&eacute; des acides nucl&eacute;iques. Apr&egrave;s la lyse de l&#39;&eacute;chantillon, l&#39;ARN est isol&eacute; et convertie en une banque d&#39;ADNc r&eacute;utilisables. Apr&egrave;s isolement d&#39;ARN, ADN est pr&eacute;cipit&eacute;e, isol&eacute;, et bisulfite de convertis pour les &eacute;tudes de m&eacute;thylation de l&#39;ADN. Nos r&eacute;sultats d&eacute;montrent la faisabilit&eacute; d&#39;isoler l&#39;ARN et l&#39;ADN d&#39;un petit nombre de cellules avec la r&eacute;p&eacute;tabilit&eacute; des r&eacute;sultats.

Isolement de l&#39;ARN et l&#39;ADN d&#39;embryons pr&eacute;implantatoires unique et un petit nombre d&#39;ovocytes chez les mammif&egrave;res pour &eacute;tudes d&#39;impression.

Ricercatori cui modelli sperimentali sono mammiferi ovociti ed embrioni preimpianto sono spesso limitati per la resa degli acidi nucleici che pu&ograve; essere isolato da una dimensione di piccolo campione. Inoltre, il numero limitato di cellule da questi tipi di campioni rende il recupero simultaneo di RNA e DNA molto difficile e spesso campione pool &egrave; necessario aumentare la resa dell&#39;acido nucleico. Qui riportiamo un semplice insieme di procedure utilizzando i kit disponibili in commercio che si traduce in costante resa e qualit&agrave; di acidi nucleici. Dopo la lisi del campione, RNA &egrave; isolato e convertito in una libreria di cDNA riutilizzabili. A seguito di isolamento del RNA, DNA &egrave; precipitato, isolato, e bisolfuro del convertito per gli studi di metilazione del DNA. I nostri risultati dimostrano la fattibilit&agrave; di isolamento di RNA e DNA da un piccolo numero di cellule con ripetibilit&agrave; dei risultati.

Md Almamun

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