Two simple and inexpensive methods for preparing DNA suitable for digital PCR from a small number of cells in 96-well plates.
96-well plates
chelex100
digital PCR
direct PCR
small number of cells
Journal
Journal of clinical laboratory analysis
ISSN: 1098-2825
Titre abrégé: J Clin Lab Anal
Pays: United States
ID NLM: 8801384
Informations de publication
Date de publication:
Jan 2021
Jan 2021
Historique:
received:
02
06
2020
revised:
08
07
2020
accepted:
10
07
2020
pubmed:
8
8
2020
medline:
21
10
2021
entrez:
8
8
2020
Statut:
ppublish
Résumé
Although DNA of high quality can be easily prepared from cultured cells with commercially available kits, many studies involve a large number of samples which increases the cost drastically. We optimized two simple and inexpensive methods for preparing DNA suitable for digital PCR from a small number of cells directly from wells of 96-well plates. Cells (number: 10 For 1000 cells from one primary culture and two tumor cell lines, DNA was reproducible and obtained with recovery rate (obtained/expected amount of DNA) in the range of 50%-90% as measured by the fluorometer dyes instrument Qubit. Using 8 out of a total of 10 µL DNA solution for 1000 cells, both conventional PCR and digital PCR were successful. For digital PCR, more than 1600 positive droplets were obtained for DNA from 1000 cells using the Direct PCR Two methods are efficient, especially the Direct PCR
Sections du résumé
BACKGROUND
BACKGROUND
Although DNA of high quality can be easily prepared from cultured cells with commercially available kits, many studies involve a large number of samples which increases the cost drastically. We optimized two simple and inexpensive methods for preparing DNA suitable for digital PCR from a small number of cells directly from wells of 96-well plates.
METHODS
METHODS
Cells (number: 10
RESULTS
RESULTS
For 1000 cells from one primary culture and two tumor cell lines, DNA was reproducible and obtained with recovery rate (obtained/expected amount of DNA) in the range of 50%-90% as measured by the fluorometer dyes instrument Qubit. Using 8 out of a total of 10 µL DNA solution for 1000 cells, both conventional PCR and digital PCR were successful. For digital PCR, more than 1600 positive droplets were obtained for DNA from 1000 cells using the Direct PCR
CONCLUSIONS
CONCLUSIONS
Two methods are efficient, especially the Direct PCR
Identifiants
pubmed: 32761657
doi: 10.1002/jcla.23513
pmc: PMC7843281
doi:
Substances chimiques
DNA
9007-49-2
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
e23513Subventions
Organisme : China Scholarship Council
ID : 201806370248
Organisme : China Scholarship Council
ID : 201806370249
Informations de copyright
© 2020 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.
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