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Genomic Services

Nucleic Acid Extraction Services

Nucleic acid extraction is one of the fundamental techniques in molecular biology and biotechnology. This involves isolation of DNA or RNA from cells and tissues. Extracted nucleic acids can be used in various applications such as PCR (polymerase chain reaction), RT-PCR (reverse transcription PCR), qPCR (quantitative PCR), DNA sequencing and various other molecular biology techniques. Following are several techniques to extract nucleic acids:

  • Non-organic method.
  • Organic method.
  • Silica based column method.
  • Magnetic bead-based extraction.

At REPROCELL (Bioserve) we provide nucleic acid extraction services for both DNA and RNA. Depending on the type of the sample, we utilize the extraction method that provides the best quality and yields of nucleic acids.

DNA Extraction

REPROCELL (Bioserve) has been providing DNA extraction services for over 30 years. We have utilized non-organic, organic, and silica-based column method based on the sample type. The sample types processed include the following:

  • Whole blood
  • Buffy coats
  • Lymphocytes
  • PBMC
  • Plasma
  • Serum
  • Blood clots
  • Blood spots
  • Solid tissues
  • Tissues from FFPE blocks
  • Saliva
  • Buccal swabs
  • Cytobrushes
  • Nasal swabs
  • Hair follicles
  • Urine
  • Feces
  • Nails
  • Tissue culture cells
  • Tissue culture media
  • Leaves
  • Roots

For many of these tissue types we use our proprietary non-organic extraction reagents, DNAQuik, that produce high quality and high molecular weight genomic DNA.

PGS blood extraction (1)PGS blood extraction (2)

Above: pouring blood into extraction tube to get them ready for centrifugation.

DNA Extraction Standard Protocol

REPROCELL (Bioserve) has developed many standard operating protocols (SOPs) for extracting DNA from tissues and other sample types. Depending on the sample type, changes are made to the SOP to obtain high quality and high molecular weight DNA. The standard protocol for extracting DNA from tissues consists of the following steps:

  • Red blood cell lysis and removal
  • Cell lysis
  • Protein removal
  • DNA precipitation
  • DNA resuspension
  • Quantification
  • Quality assessment
  • Aliquoting
  • Storage
  • Shipping

PGS blood lysis solutionPGS blood lysis (2)

Above: Adding blood lysis solution and vortexing the tubes

DNA Extraction from blood clots

The protocols for extracting DNA from blood clots requires a breakdown of the clot using a proprietary clot breaking device called a “Clot Bucket”.   The device is placed in a 50 ml conical centrifuge as shown in the figure and the blood clot is poured into the clot bucket along with the DNAQuik “Blood Lysis Buffer” and centrifuged at 4000 RPM for 20 minutes, this breaks the blood clot down into small pieces that facilitates the DNA extraction using the SOP.

PGS - DNA extraction clots

A – Clot Bucket
B – Inserting clot bucket into a 50ml centrifuge tube
C – Pouring blood clot into the inserted clot bucket in the tube
D – Loading the tube with clot bucket and clot in a centrifuge

DNA Extraction from tissues from FFPE tissue blocks

To extract DNA from FFPE tissue blocks, we first cut 5- to 10-micron sections from the block. These sections are transferred to a 15 ml tube. The protocol we use is a combination of organic and non-organic methods. Following are the steps involved in this process:

  • Sample de-paraffinization using organic solvents
  • Sample lysis
  • Protein removal
  • DNA precipitation
  • DNA resuspension
  • DNA quantitation using RT- PCR

Above: FFPE block.

PGS amplification plot (3)

Above: RT-PCR analysis of DNA extracted from FFPE tissues

DNA Extraction from frozen solid tissues

The frozen solid tissues are first ground to a powder using mortar and pestle under liquid nitrogen to keep the integrity of DNA/RNA. This powdered tissue is then transferred to a 50 ml conical tube and processed using the standard DNA extraction protocol. Concentration and quantity of DNA is determined by measuring UV absorbance at 260 nm. The 260 nm / 280 nm ratio is used to assess the purity of DNA, which should be between 1.7 and 1.9.  The quality of DNA can also be determined by electrophoresing DNA on a 1% agarose gel along with standard molecular weight markers.

PGS extracted DNA (electrophoresis)

Above: DNA extracted from solid tissues electrophoresed on a 1% agarose gel along with standard molecular weight markers.

DNA Extraction from plasma and serum

REPROCELL (Bioserve) uses specialized column-based kits for extracting DNA from plasma and serum.  We follow the protocol supplied by the manufacturer of the extraction kits.  DNA is assessed for quality and quantity by RT-PCR.  We routinely extract ctDNA from plasma for NGS analysis. 

Above: RT-PCR analysis of ctDNA extracted from plasma of cancer subjects along with standards to determine quality and concentration of DNA

Above: standard curve was used to estimate the quantity and concentration of ctDNA 

DNA extraction from saliva

REPROCELL (Bioserve) has extracted DNA from several thousand saliva samples. We use non-organic, DNAQuik, reagents for this process. The typical protocol is as follows:  

  • Pour the saliva in to 50 ml conical tube
  • Centrifuge at 4000 RPM to pellet cells
  • Sample lysis
  • Protein removal
  • DNA precipitation
  • DNA resuspension
  • DNA quantitation using RT- PCR

RNA Extraction

REPROCELL has 20 years’ experience with RNA isolation from a variety of starting materials. We have been successful in RNA extraction from whole blood, buffy coats, PAXgene tubes, lymphocytes, cultured cells, plant material, cartilage, fresh and frozen tissues and various bacteria.

REPROCELL utilizes reagents from Qiagen and/or RNAQuikTM reagents depending on the type of tissue. REPROCELL has developed protocols for RNA purification from tissues such as cartilage.

REPROCELL will provide A260/A280 ratio and concentration for RNAs isolated. The quality of RNA isolated is determined using Agilent’s Bioanalyzer. RNA isolated at REPROCELL has been used in various molecular biology and genomics applications. These applications include expression analysis using RT-PCR, micro array analysis using Affymetrix GeneChip system and library construction.

To obtain high quality RNA, REPROCELL recommends that the samples should be immediately cryo-preserved upon collection and properly shipped to REPROCELL. Sample sets received for RNA extraction are logged in, entered into a database, and stored at −70°C until processing. After RNA extraction is completed, samples are frozen at −70°C, packaged with a data sheet and Bioanalyzer data, and shipped to the customer.

Nucleic Acid Extraction Services

Case Study: DNA Extraction from Whole Blood

REPROCELL(Bioserve) has been providing DNA extraction from whole blood for a government institution since 1995. Under this contract the customer sends 500 whole blood samples (10 ml) once every three weeks. REPROCELL (BioServe) personnel extract, quantify, quality assess, aliquot and ship the DNA back to the customer’s repository. The processes involved in this project are detailed below. 

PGS prepared labeled tubes
50 ml tubes labeled and ready for processing whole blood samples

The steps for extracting DNA from 6 ml of whole blood vary slightly from the above outlined steps. First the red blood cells must be lysed and removed. To accomplish this at REPROCELL(Bioserve) we use DNAquik Red Blood Cell lysis buffer. This is followed by the steps below:

  • Lysis of white blood cells
    • This is accomplished by using our proprietary DNAQuik “Sample Lysis” solution consisting of detergents and enzymes to release nucleic acids and proteins.

PGS blood lysis solution (2)
Adding “Sample Lysis” solution to white blood cells

  • Protein and lipid removal
    • Proteins and lipids are removed using our proprietary DNAQuik “Protein Out” solution consisting of high salt concentration.
  • DNA precipitation
    • DNA is precipitated using isopropanol at an appropriate ratio.
  • DNA resuspension
    • DNA is resuspended in 100 µl of DNAQuik “DNA Resuspending Buffer” overnight on a shaker at room temperature.

PGS - DNA resuspension shaker

DNA resuspension at room temperature

  • Quantification
    • DNA is quantified by UV spectrophotometry. DNA absorbs UV light at 260 nm and the concentration can be determined by measuring absorbance at this wavelength. Nanodrop UV spectrophotometer scans the DNA sample from 220 nm to 300 nm for assessing the quality of DNA.  

PGS Nanodrop equipment

Measuring absorbance using Nanodrop UV spectrophotometer

PGS Nanodrop display screen

Absorbance read-out of DNAs using a Nanodrop UV spectrophotometer

  • Quality assessment
    • The ratio of absorbance of DNA at 260 nm to that at 280 nm (A260/A280) is used to assess the quality/purity of DNA. A ratio of 1.7 to 1.9 indicates pure DNA. The table below illustrates the quality assessment of the extracted DNA.

BBLID A260 A280 RATIO Concentration (ng/µl) Volume µ(l) Yield (µg)
DNA2304-1 2.46 1.36 1.81 123.15 175 21.55
DNA2304-2 1.76 1.00 1.77 88.10 175 15.42
DNA2304-3 1.15 0.64 1.80 57.55 175 10.07
DNA2304-4 0.93 0.52 1.78 46.40 175 8.12
DNA2304-5 0.91 0.48 1.87 45.35 175 7.94
DNA2304-6 0.53 0.28 1.88 26.55 175 4.65
DNA2304-7 2.63 1.43 1.84 131.55 175 23.02
DNA2304-8 1.44 0.79 1.83 71.85 175 12.57
DNA2304-9 0.94 0.54 1.75 46.80 175 8.19
DNA2304-10 1.38 0.76 1.81 68.75 175 12.03
  • Normalization
    • All the DNAs are normalized to a concentration of 250 ng/µl.
  • Storage
    • The DNAs are stored at 4 °C until aliquoted.
  • Aliquoting
    • REPROCELL(Bioserve) provides custom aliquoting based on customers’ requirements. For this project, customer requires 4 aliquots of 10 µg at 100 ng per µl, 3 aliquots of 50 µg at 250 ng per µl and remaining DNA in three equal aliquots. All these tubes must be 2D barcoded with a bottom 2D barcode. Once the aliquoting is done, the bottom barcode and the side barcode must be read to confirm the identity of DNA. Approximately 3000 aliquots are made for each batch of 500 whole blood samples.

PGS labeling aliquots

Above: Labeling the aliquot tubes

PGS aliquot racks

  • Shipping
    • Once all the data is checked and confirmed by the project leader DNA samples are shipped back to the customer designated repository via FedEx in dry ice.