Sample QC

Question 1

how does the nano drop work?

Answer 1

its a spectrophotometer that measures the light absorbed by a solution: Absorbance

Question 2

what information does the Nanodrop provide you with?

Answer 2

• Purity, DNA/RNA (contaminations)

- concentration (ng/uL)

Question 3

what information can’t the Nanodrop provide?

Answer 3

• integrity of the sample (Degradation)

- can’t tell if RNA id contaminated with DNA and vice versa

Question 4

what does the absorbance ratio at 260/280nm tell you

Answer 4

highlights the possible contamination by proteins

Question 5

what does the absorbance ratio at 260/230nm tell you

Answer 5

highlights the possible contamination by salt, carbs, peptides and phenol

Question 6

what is considered the optimal ratio for DNA and RNA at 260/280nm

Answer 6

DNA 1.8 - 2.0

RNA 2.0 - 2.2

Question 7

what is considered the optimal ratio for DNA and RNA at 260/230nm

Answer 7

DNA 2.0-2.2

RNA 2.0 - 2.2

Question 8

what is the reason for the optimal ratio of DNA being lower than that of RNA at 260/280

Answer 8

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

Question 9

at what wavelength does protein absorb UV light

Answer 9

280nm

Question 10

at what wavelength do salts, carbs, peptides and phenol absorb UV light

Answer 10

230nm

Question 11

what is the concentration range of the nano drop

Answer 11

25-2000ng/uL

Question 12

what equation is used to convert A260 values into ng/uL

Answer 12

beer-lambert law

Question 13

how can a sample be determined to be DNA or RNA using the A260 value and the beer-lambert law

Answer 13

when A260 = 1 then:

• the constant will be 50 if its DNA
• the constant will be 40 if its RNA

Question 14

On the absorbance plot for the nano drop, what relationship should the 280 and 230 peaks have with the 260 peak

Answer 14

the 280 and 230 peak should be half the hight of the 260 peak

Question 15

what wavelength do DNA and RNA absorb UV light at

Answer 15

260nm

Question 16

what are the benefits of using nanodrop

Answer 16

quick and affordable

only needs small amount of sample i.e. 25ng/uL

Question 17

how is the RIN calculated

Answer 17

the ratio created by putting the 28S peak over the 18S peak

Question 18

why is nano drop so important when QCing in studies

Answer 18

its the only way to assess purity and contamination in DNA and RNA

Question 19

what is the Qubit

Answer 19

a quantification method that uses fluorescent dyes that bind to DNA/RNA molecules which then emit light when excited

Question 20

what is the concentration range of the Qubit

Answer 20

DNA 2-1000ng/uL

RNA 20-1000ng/uL

Question 21

how is the qubit sample prepared

Answer 21

mixed with the dye and kit buffer in an eppendorf

Question 22

what information is gained from the Qubit

Answer 22

sample concentration

- fluorescence values converted into conc,

Question 23

benefits of Qubit?

Answer 23

• more sensitive and accurate

- good for PCR and NGS as it already contains the fluorescent tags to be amplified

Question 24

what information is not provided by Qubit

Answer 24

no info on purity

no info on integrity

Question 25

what is the mechanism of the tapestation

Answer 25

it performs automated electrophoresis in an agarose gel which separates molecules based on their size and charge using an electrically charged field

Question 26

what charge do DNA and RNA molecules have

Answer 26

negative

Question 27

what constitutes the RNA analysis

Answer 27

RNA ladder RNA buffer RNA sample RNA screentape

Question 28

what is the RNA ladder

Answer 28

a set of different and known RNAs band size used as a reference for estimating size and mass of an RNA aample

Question 29

what type of RNA is used as the assessment of RNA

Answer 29

Ribosomal RNA (rRNA)

Question 30

what is the RIN

Answer 30

A numerical value between 1 and 10 used to indicate RNA integrity

Question 31

what is the threshold RIN value for an RNA sample to be deemed to have high enough integrity and suitable for downstream applications?

Answer 31

7

Question 32

how are quantity and integrity derived form the tapestation/bioanalyzer graph

Answer 32

concentration is derived from the height of the peak (quantity) size of fragments is based on width of the peak (integrity)

Question 33

what information does tapestation/bioanalzyer give you

Answer 33

- integrity of DNA/RNA - Info on size, quantity and quality - shows whether there is a DNA or RNA contamination

Question 34

what information about contamination can the bioanalyzer tell you that nano drop can't?

Answer 34

whether DNA is contaminated with RNA and vice versa

Question 35

what information can't the bioanalyzer tell you

Answer 35

no information about purity other than DNA and RNA contaminations

Question 36

why is the tapestation better than other integrity analysis methods like gel electrophoresis?

Answer 36

because gel electrophoresis only allows you to visually assess purity however tapestation offers an objective RIN value that uses a standardised equation/procedure.

Question 37

how is DNA concentration determined using the bioanalyzer graph

Answer 37

using the area under the peak compared to the known conc of the upper marker