Scribd Logo
Upload

Welcome to Scribd!

  • QC - Tapestation - Amended For COG-train

    Uploaded by

    Carlos Ramírez
    11 views14 pages
    1. The Tapestation is an automated electrophoresis system that separates biomolecules like DNA and RNA by size and charge to analyze their size, concentration, and integrity. 2. It uses a gel, buffer chamber, and electrodes to separate nucleic acids run through 16 channels. RNA analysis examines ladder, buffer, sample, and screentape to assess integrity using the 18S and 28S ribosomal RNA peaks. 3. DNA analysis similarly examines ladder, buffer, sample, and screentape to determine concentration from peak height and integrity from peak width and shape. The Tapestation provides quality control data but not purity assessment.

    Original Description:

    Original Title

    QC_-_Tapestation_-amended_for_COG-train.pptx

    Copyright

    © © All Rights Reserved

    Available Formats

    PDF, TXT or read online from Scribd

    Did you find this document useful?

    Is this content inappropriate?

    Report this Document
    1. The Tapestation is an automated electrophoresis system that separates biomolecules like DNA and RNA by size and charge to analyze their size, concentration, and integrity. 2. It uses a gel, buffer chamber, and electrodes to separate nucleic acids run through 16 channels. RNA analysis examines ladder, buffer, sample, and screentape to assess integrity using the 18S and 28S ribosomal RNA peaks. 3. DNA analysis similarly examines ladder, buffer, sample, and screentape to determine concentration from peak height and integrity from peak width and shape. The Tapestation provides quality control data but not purity assessment.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Flag for inappropriate content
    11 views14 pages

    QC - Tapestation - Amended For COG-train

    Uploaded by

    Carlos Ramírez
    1. The Tapestation is an automated electrophoresis system that separates biomolecules like DNA and RNA by size and charge to analyze their size, concentration, and integrity. 2. It uses a gel, buffer chamber, and electrodes to separate nucleic acids run through 16 channels. RNA analysis examines ladder, buffer, sample, and screentape to assess integrity using the 18S and 28S ribosomal RNA peaks. 3. DNA analysis similarly examines ladder, buffer, sample, and screentape to determine concentration from peak height and integrity from peak width and shape. The Tapestation provides quality control data but not purity assessment.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Flag for inappropriate content
    of 14

    Quality Control

    Tapestation
    Dr Paola Niola FHEA
    Senior Applications Specialist
    Teaching Fellow
    UCL Genomics
    Learning outcomes

    You will learn:

    1. How the Tapestation works


    2. How to interpret Quality Control (QC) data
    Tapestation
    • Automated electrophoresis system which separates charged biomolecules
    in a fluid using a field electrically charged

    • Analysis of SIZE, CONCENTRATION and INTEGRITY

    Screentape

    • 16 channels: buffer chamber, gel, electrodes


    • DNA and RNA (negative charged) are separated by size and weight
    RNA Tapestation analysis

    1. RNA Ladder
    2. RNA buffer
    3. RNA sample
    4. RNA screentape
    RNA
    Ladder

    The RNA ladder is a set of different and known RNAs band size.

    The RNA ladder is a reference for estimating size and mass of RNA sample.
    RNA
    Quantification

    • Lower marker: 25nt fragment, run with each of the


    samples, internal marker used to align all samples
    to the ladder data. Concentration is calculated considering
    the area under the 18S and 28S peaks
    • 18S and 28S peaks of Eukaryote total RNA: compared to the area under the ladder.
    - 80% of tot RNA
    - used to assess the quantity & quality.
    RNA
    Integrity
    Ribosomal RNA is used as assessment of RNA integrity

    The RNA integrity number (RIN):

    • Numerical value to indicate the RNA integrity


    Scale 1-10 (10 being perfect)
    Higher the number, better the quality of RNA sample is!

    • The 28S/18S ratio gives an indication on the RNA integrity


    RNA Tapestation
    examples
    RNA gel
    Ladder

    Lower
    marker

    28S
    28S
    18S
    5S 18S
    Good trace!
    5S
    Lower
    marker
    RNA
    Integrity
    Number RNA with RIN equal or > 7 is suitable for downstream applications!
    (RIN)
    Ladder

    Degraded RNA!
    Lower
    marker
    RNA
    Integrity
    Number
    (RIN)
    DNA Tapestation analysis

    1. DNA Ladder
    2. DNA buffer
    3. DNA sample
    4. DNA screentape
    DNA
    Ladder

    The DNA ladder is a set of DNAs of known size, important to estimate size and mass of DNA sample.

    Lower marker is an internal standard used to align the ladder data to all samples.
    DNA
    Quantification
    & C
    Integrity

    A E
    B

    A (y axis) the fluorescence intensity D concentration based on the height of the peak: Quantity!
    B (x axis) size in basepair E size of fragments based on the width of the peak: Integrity!
    C Lower marker DNA Integrity Number (DIN)
    Tapestation
    Advantage:
    • The best method to assess integrity of DNA and RNA
    • RNA Integrity Number (RIN) or DNA Integrity Number (DIN)
    • Information about size, quantity and quality (integrity)

    Limitation:
    • No information about purity (use the NanoDrop instead!!!)

    Combine more methods to have an accurate and


    complete picture of your sample quality!

    You might also like