Extraction of nucleic acid and cDNA synthesis
Extraction and purification of RNA, miRNA, DNA, and protein
TATAA Biocenter has extensive experience of extraction of nucleic acids (RNA, miRNA, DNA) from a wide range of samples types deriving from a great variety of organisms and is the Swedish representative in the European consortium SPIDIA that develops guidelines for the preanalytical steps in biomolecular analysis. Extracting nucleic acids of high quality is essential for any downstream analysis. Different sample matrices require different extraction procedures and our instrument park has several options for optimal and time-effective sample preparation. Our ready-to-go protocols for extraction of nucleic acids from a broad range of different sample types enable a short start-up phase.
Samples types deriving from a variety of organisms that have already successfully processed at TATAA Biocenter include blood (including PAXgene tubes), yeast, plants, bacteria, cartilage pellets, bone, tumors, biopsies bone marrow and colon caps, saliva, formalin fixed paraffin embedded (FFPE) tissues, cervical samples, LMD samples, stem cells, single cells, process samples for residual DNA, etc. We routinely extract nucleic acids from tissues (including biopsies) where common tissues are liver, spleen, heart, brain, kidney, and colon. We also work with cell pellets, cultures, and single cells. The cells and tissues are most commonly either snap frozen, in RNALater or trizol, or positioned on different kinds of surfaces. If your sample types do not already have a validated extraction protocol, use our expertise to set up a protocol optimized for quality nucleic acids with high yield and purity.
In combination with the extraction quantity testing is performed. All extracted samples are measured for concentrations and for purity by 260/280- and 260/230- ratios on NanoDrop ND-1000 (see below) and further quality testing is also available.
Quantity and quality testing of RNA, miRNA, DNA, and protein
TATAA Biocenter has access to many methods and instruments for quantifying the amounts of biomolecules and testing samples for quality, integrity and purity. This provides highest quality and reproducibility of protocols, which is essential for any downstream processing and analyses.
We offer to analyse the quantity and quality of you nucleic acids and protein. Our systems require minute sample volumes (less than 2 µl), making sure you will have the most of your sample present for further analyses. Our platforms for automated gel electrophoresis make your analyses fast and highly cost efficient and nucleic acids can also be quantified using NanoDrop ND-1000 as well as PicoGreen.
Residual DNA analysis
TATAA Biocenter offers sensitive quantification of residual DNA from E. Coli, S. Cerevisiae, P. pastoris, Chinese hamster ovary cells, or other organisms used in your expression system. Real-time PCR is used to compare the amount of DNA in your sample to a standard curve generated from known amounts of host cell DNA.
- Choice and optimization of nucleic acid extraction protocol
- Assay design and validation for selected gene and internal control DNA
- Production of standard curves from host cell DNA
- Determination of limit of detection (LOD) and limit of quantification (LOQ) values
- Real-time PCR analysis of standards, unknown samples, and controls
Normalization and cDNA synthesis
RNA needs to be converted into cDNA for real-time PCR analysis with reverse transcription (RT). Our experts, who laid the ground for all optimization protocols of reverse transcription (Clinical Chemistry 50:3 509–515, 2004), will provide you with the best method possible. To avoid variations in the amounts of sample used for the RT, a normalization of the RNA concentrations should be performed before the cDNA synthesis.