Sample QC Flashcards
how does the nano drop work?
its a spectrophotometer that measures the light absorbed by a solution: Absorbance
what information does the Nanodrop provide you with?
- Purity, DNA/RNA (contaminations)
- concentration (ng/uL)
what information can’t the Nanodrop provide?
- integrity of the sample (Degradation)
- can’t tell if RNA id contaminated with DNA and vice versa
what does the absorbance ratio at 260/280nm tell you
highlights the possible contamination by proteins
what does the absorbance ratio at 260/230nm tell you
highlights the possible contamination by salt, carbs, peptides and phenol
what is considered the optimal ratio for DNA and RNA at 260/280nm
DNA 1.8 - 2.0
RNA 2.0 - 2.2
what is considered the optimal ratio for DNA and RNA at 260/230nm
DNA 2.0-2.2
RNA 2.0 - 2.2
what is the reason for the optimal ratio of DNA being lower than that of RNA at 260/280
because uracil has a higher absorbance ratio than thymine when measured individually causing the base absorbance of RNA to be naturally slightly higher than that of DNA
at what wavelength does protein absorb UV light
280nm
at what wavelength do salts, carbs, peptides and phenol absorb UV light
230nm
what is the concentration range of the nano drop
25-2000ng/uL
what equation is used to convert A260 values into ng/uL
beer-lambert law
how can a sample be determined to be DNA or RNA using the A260 value and the beer-lambert law
when A260 = 1 then:
- the constant will be 50 if its DNA
- the constant will be 40 if its RNA
On the absorbance plot for the nano drop, what relationship should the 280 and 230 peaks have with the 260 peak
the 280 and 230 peak should be half the hight of the 260 peak
what wavelength do DNA and RNA absorb UV light at
260nm
what are the benefits of using nanodrop
quick and affordable
only needs small amount of sample i.e. 25ng/uL
how is the RIN calculated
the ratio created by putting the 28S peak over the 18S peak
why is nano drop so important when QCing in studies
its the only way to assess purity and contamination in DNA and RNA
what is the Qubit
a quantification method that uses fluorescent dyes that bind to DNA/RNA molecules which then emit light when excited
what is the concentration range of the Qubit
DNA 2-1000ng/uL
RNA 20-1000ng/uL
how is the qubit sample prepared
mixed with the dye and kit buffer in an eppendorf
what information is gained from the Qubit
sample concentration
- fluorescence values converted into conc,
benefits of Qubit?
- more sensitive and accurate
- good for PCR and NGS as it already contains the fluorescent tags to be amplified
what information is not provided by Qubit
no info on purity
no info on integrity
what is the mechanism of the tapestation
it performs automated electrophoresis in an agarose gel which separates molecules based on their size and charge using an electrically charged field
what charge do DNA and RNA molecules have
negative
what constitutes the RNA analysis
RNA ladder
RNA buffer
RNA sample
RNA screentape
what is the RNA ladder
a set of different and known RNAs band size used as a reference for estimating size and mass of an RNA aample
what type of RNA is used as the assessment of RNA
Ribosomal RNA (rRNA)
what is the RIN
A numerical value between 1 and 10 used to indicate RNA integrity
what is the threshold RIN value for an RNA sample to be deemed to have high enough integrity and suitable for downstream applications?
7
how are quantity and integrity derived form the tapestation/bioanalyzer graph
concentration is derived from the height of the peak (quantity)
size of fragments is based on width of the peak (integrity)
what information does tapestation/bioanalzyer give you
- integrity of DNA/RNA
- Info on size, quantity and quality
- shows whether there is a DNA or RNA contamination
what information about contamination can the bioanalyzer tell you that nano drop can’t?
whether DNA is contaminated with RNA and vice versa
what information can’t the bioanalyzer tell you
no information about purity other than DNA and RNA contaminations
why is the tapestation better than other integrity analysis methods like gel electrophoresis?
because gel electrophoresis only allows you to visually assess purity however tapestation offers an objective RIN value that uses a standardised equation/procedure.
how is DNA concentration determined using the bioanalyzer graph
using the area under the peak compared to the known conc of the upper marker
