We use cookies to understand how you use our site and to improve your experience. This includes personalizing content and advertising. To learn more, click here. By continuing to use our site, you accept our use of cookies. Cookie Policy.

Features Partner Sites Information LinkXpress hp
Sign In
Advertise with Us

THERMO FISHER SCIENTIFIC

Thermo Fisher Scientific provides analytical instruments, lab equipment, specialty diagnostics, reagents and integrat... read more Featured Products: More products

Download Mobile App




Two Simple Methods Prepare DNA Suitable For Digital PCR

By LabMedica International staff writers
Posted on 19 Aug 2020
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.

In addition, a limited amount of each sample is often a challenge, for example, for studies using cells of primary cultures for testing multiple drugs at multiple concentrations in multiple replicates, and studies or diagnosis involving defined subpopulations of human immune cells.

Medical scientists at the University Hospital Hamburg‐Eppendorf (Hamburg, Germany) used cells from a primary culture derived from a benign plexiform neurofibroma, a melanoma cell line, and a human fibroblast line. More...
The cells were seeded into wells of 96‐well plates with at different concentrations, each in three replicates. The plates were incubated overnight for the cells to attach the cultural surface and subjected to DNA extraction on the next day after checking the adhesive living cells under a microscope.
After removing the medium, the wells were washed twice with PBS. Subsequently, 70 μL of the Direct PCR lysis reagent (PeqLab, Erlangen, Germany) supplemented with 0.2 mg/mL fresh proteinase K together with 70μL water was added to each well containing adhesive living cells. Supernatants containing the extracted DNA were further purified by precipitation. The team also lysed cells using chelex100 powder (Bio-Rad, Hercules, CA, USA). Conventional polymerase chain reaction (PCR) was carried out using 2 µL out of the 10 µL DNA and a primer pair for an exon of the NF1 gene which is used for the routine genetic diagnosis in their laboratory.

The investigators reported that for 1,000 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 (Thermo Fisher Scientific, Waltham, MA, USA). For digital PCR, more than 1,600 positive droplets were obtained for DNA from 1,000 cells using the Direct PCR method, corresponding to a yield efficiency of approximately 80%. Further reducing the number of cells down to 100 would be possible with 160 positive droplets expected. Both reagents are inexpensive at EUR 0.08/sample.

The authors concluded that the two methods were efficient; especially the Direct PCR reagent‐based method provides a simple and inexpensive method for preparing DNA suitable for digital PCR from small number of cells. The study was published on August 5, 2020 in the Journal of Clinical Laboratory Analysis.

Related Links:

University Hospital Hamburg‐Eppendorf
PeqLab
Bio-Rad
Thermo Fisher Scientific

New
Gold Member
Hybrid Pipette
SWITCH
Portable Electronic Pipette
Mini 96
8-Channel Pipette
SAPPHIRE 20–300 µL
Sample Transportation System
Tempus1800 Necto
Read the full article by registering today, it's FREE! It's Free!
Register now for FREE to LabMedica.com and get access to news and events that shape the world of Clinical Laboratory Medicine.
  • Free digital version edition of LabMedica International sent by email on regular basis
  • Free print version of LabMedica International magazine (available only outside USA and Canada).
  • Free and unlimited access to back issues of LabMedica International in digital format
  • Free LabMedica International Newsletter sent every week containing the latest news
  • Free breaking news sent via email
  • Free access to Events Calendar
  • Free access to LinkXpress new product services
  • REGISTRATION IS FREE AND EASY!
Click here to Register








Channels

Hematology

view channel
Image: New evidence shows viscoelastic testing can improve assessment of blood clotting during postpartum hemorrhage (Photo courtesy of 123RF)

Viscoelastic Testing Could Improve Treatment of Maternal Hemorrhage

Postpartum hemorrhage, severe bleeding after childbirth, remains one of the leading causes of maternal mortality worldwide, yet many of these deaths are preventable. Standard care can be hindered by delays... Read more

Immunology

view channel
Image: The CloneSeq-SV approach can allow researchers to study how cells within high-grade serous ovarian cancer change over time (Photo courtesy of MSK)

Blood Test Tracks Treatment Resistance in High-Grade Serous Ovarian Cancer

High-grade serous ovarian cancer (HGSOC) is often diagnosed at an advanced stage because it spreads microscopically throughout the abdomen, and although initial surgery and chemotherapy can work, most... Read more

Industry

view channel
Image: The collaboration aims to improve access to Hb variant testing with the Gazelle POC diagnostic platform (Photo courtesy of Hemex Health)

Terumo BCT and Hemex Health Collaborate to Improve Access to Testing for Hemoglobin Disorders

Millions of people worldwide living with sickle cell disease and other hemoglobin disorders experience delayed diagnosis and limited access to effective care, particularly in regions where testing is scarce.... Read more
Copyright © 2000-2025 Globetech Media. All rights reserved.