illustration of people in lab coats working on dna

PCR

Cell line development (CLD)

Despite advancements in CRISPR and other cell line development technologies, the path from starting cell to a modified one remains long and tricky. What if there is a way to improve and accelerate your cell line development workflow while uncovering any unintended changes to the genome?
The Insight Exchange on Cell Line Development
Find out the best practices in cell line development and cell and gene therapy from renowned global scientists
The vulnerabilities of cell line engineering
  • Given the low success rate of gene editing techniques, screening for successfully edited clones and characterizing the edits could take several months to complete. We are here to make your cell line development process easier and faster.
  • Gene editing, viral transduction or even routine transfections can cause off-target effects such as chromosomal rearrangements and structural variants, often missed by classical methods like karyotyping and FISH. Choosing your genome assessment method wisely when working with cell line development is indeed an important decision in getting the cell right. 
One week for:
One week for:
  • Transfection
  • Transduction
  • CRISPR gene editing
Several months for:
Several months for:
  • Clone selection
  • Sanger sequencing
  • qPCR validation
  • Gene expression analysis
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Several weeks for:
Several weeks for:
  • Genome integrity assessment
  • Off-target analysis
  • RNA sequencing
Explore products
Ready to accelerate your clone selection workflow?
Go to our dedicated page to explore the QIAprep&amp CRISPR kit and QuantiNova one-step RT-qPCR solutions in detail.
Don’t sacrifice speed for quality – instead, combine both in your clone selection and cell line characterization workflow
Is CRISPR screening keeping you occupied for weeks?

Slash unnecessary tasks from your CRISPR gene edit characterization. Watch the video to learn how.


Analyze gene expression directly from cells and make your edits faster and easier
Take a 3X faster route to your clone of interest

Characterize CRISPR gene edits in 3 days using only 10 cells/µL. 

hands in blue gloves marking petri dish
Gene expression analysis – validate gene edits at the transcript level using QuantiNova PCR
person working in lab
Perform qPCR directly from cells

Minimize the time and effort needed to identify your clone of interest. Streamlining your cell culture and gene editing process can help you obtain the desired clone early on.

Reveal the true genome status within 5 days

Watch this video to discover a simple yet elegant approach to look for unintended genome changes in your engineered cells*.

Identify chromosomal alterations early on during the cell line development process
*Solely for the US: No rights to use this product to perform or offer any service in metagenomics or structural variation analysis are granted by the supply of this product, whether expressly, by implication or by estoppel.
Why choose EpiTect Hi-C over karyotyping or FISH? 

Compared to classic methods,  EpiTect Hi-C gives you a 10X sharper view into the genome, uncovering genomic blind spots such as chromosomal rearrangements and structural variants*.  Learn more in this flyer.  

beware of blind spots illustration
*Solely for the US: No rights to use this product to perform or offer any service in metagenomics or structural variation analysis are granted by the supply of this product, whether expressly, by implication or by estoppel.
Get ahead of unintended changes lurking in your cells
Go to our dedicated page and explore EpiTect Hi-C

FAQs

CRISPR screening
Is there anything to consider when processing cultured cells on coated cultivation dishes? Are additional purification steps required to eliminate or reduce coating reagents such as Poly-L-Lysine?
The QIAprep&amp CRISPR sample preparation and target amplification are not affected by coating reagents. Additional information and a list of tested coating reagents can be found in the QIAprep&amp CRISPR and CRISPR-Q Custom Kits Handbook in Appendix C.
Is there something to consider when working with transduced cells treated with Polybrene?
The AllTaq PCR chemistry included in the QIAprep&amp CRISPR Kit is very powerful against inhibitors. The sample preparation is not affected, and the PCR reaction tolerates up to 1 µg/mL.
Can the new CRISPR PCR Assays be used for digital PCR (dPCR) on the QIAcuity? Do you have data or a protocol?
The CRISPR-Q Custom PCR Assay is not for dPCR use. It is for a conventional PCR run in a standard cycler, and the PCR product is analyzed on the QIAxcel or a gel. The CRISPR-Q Custom PCR Assay contains 2 primers flanking the cut site. The purpose is to do a quick PCR check of the clones as well as preparation of template for the following Sanger sequencing. For Sanger sequencing, the corresponding CRISPR-Q Sanger Primers are used. dPCR for checking the editing event is an alternative to Sanger Sequencing and would be specific for the event.
EpiTect Hi-C
Are there stopping points in the EpiTect Hi-C workflow?
Yes. Following the Hi-C Digestion, Hi-C End Labeling, Hi-C Ligation, or Chromatin De-crosslinking steps in the Hi-C Part 1 workflow, users can store samples at −15 to −25°C and resume with the procedure at a later date.
What is the effect of using amounts of input material per sample that fall outside the recommended range?
Using input amounts outside the range of 5 x 103 and 2.5 x 106 human or a mouse cell (or the equivalent of 30 ng–15 µg DNA) per sample risks affecting the quality of the resulting next-generation sequencing (NGS) library, such that it is likely to have higher levels of inward-facing (FR) sequence read bias, un-ligated ends, and read pairs containing contiguous, re-ligated restriction fragments.
Can I use cells that have been previously crosslinked and frozen (e.g., for a ChIP or ChIP-seq experiment) as input material for the EpiTect Hi-C protocol?
Yes. However, the cells should have been crosslinked with 1% formaldehyde and not have been frozen for longer than 6 months.
With which platform can I perform my sequencing e.g. within cell line development process (CLD)?
EpiTect Hi-C sequencing libraries are compatible with all Illumina sequencing platforms.
Can I use my own pipeline to analyze results from my EpiTect Hi-C experiments e.g. in cell line development (CLD)?
Yes. For your analysis, you will need the following additional information. The endonuclease used in the EpiTect Hi-C Kit cuts at GATC sites. The Hi-C junction motifs (or tags) generated after Hi-C ligation consist of a GATCGATC sequence.
With which read length should the EpiTect Hi-C NGS libraries be sequenced?
Read lengths of 36–150 bp can be used to sequence the EpiTect Hi-C libraries on Illumina sequencers. However, as mappability increases with read length, we suggest to use 2 x 150 bp sequencing reads for best results.
Does QIAGEN provide data analysis to accompany the EpiTect Hi-C Kit?
Yes. Visit QIAGEN’s online GeneGlobe Data Analysis Center at Hi-C Analysis (qiagen.com) to analyze your data with the EpiTect Hi-C Data Analysis Portal.
QuantiNova RT-PCR kit
Do I require any specific equipment to analyze a cultured cell after direct lysis using QuantiNova RT-qPCR Kits?
Standard laboratory equipment and real-time cyclers are suited for this procedure. To select suitable assays, visit QuantiNova LNA Assays via GeneGlobe for SYBR Green or Probe-based detection of mRNA.
How many cells are required to check gene expression levels of my edited gene(s) in direct PCR?
QuantiNova one-step RT-qPCR kits can detect mRNA even from a single cell. However, researchers should consider the expected abundance of the edited genome of interest to select an appropriate amount of cells. Up to 2000 cells per PCR reaction vessel are recommended.
Which cell lines are suited for direct lysis and RT-qPCR?
Various cell lines were successfully used in the described direct PCR process, and all commonly used cell lines should likely be suited for this procedure.
Is it required to wash cells before direct lysis and RT-qPCR?
Yes, both extracellular and debris and intracellular material from dead cells, including RNA or RNases and proteases, will be released in the cell culture and can interfere with RT-PCR. We strongly recommend removal by washing them with a cell culture medium.
Do I need to set up the RT-PCR reaction on ice?
No, QuantiNova qRT-PCR Kits use a two-phase one-step qRT-PCR that keeps both reverse transcriptase and Taq polymerase inactive at ambient temperature, allowing convenient room-temperature setup without compromising performance.