Respiratory Viruses Identification Lab

RNA Extraction

1. Pipet 560 μl prepared Buffer AVL containing carrier RNA into a 1.5 ml microcentrifuge
tube.

2. Add 140 μl plasma, serum, urine, cell-culture supernatant or cell-free body fluid to the
Buffer AVL–carrier RNA in the microcentrifuge tube. Mix by pulse-vortexing for 15 s.

3. Incubate at room temperature (15–25°C) for 10 min.

Note: Viral particle lysis is complete after lysis for 10 min at room temperature. Longer
incubation times have no effect on the yield or quality of the purified RNA.

4. Briefly centrifuge the tube to remove drops from the inside of the lid.

5. Add 560 μl ethanol (96–100%) to the sample, and mix by pulse-vortexing for 15 s. After
mixing briefly centrifuge the tube to remove drops from inside the lid.
6. Carefully apply 630 μl of the solution from step 5 to the QIAamp Mini column (in a 2 ml
collection tube) without wetting the rim. Close the cap, and centrifuge at 6000 x g
(8000 rpm) for 1 min. Place the QIAamp Mini column into a clean 2 ml collection tube,
and discard the tube containing the filtrate.
Note: Close each spin column to avoid cross-contamination during centrifugation.
Note: Centrifugation is performed at 6000 x g (8000 rpm) to limit microcentrifuge noise.
Centrifugation at full speed will not affect the yield or purity of the viral RNA. If the
solution has not completely passed through the membrane, centrifuge again at a higher
speed until all the solution has passed through.

7. Carefully open the QIAamp Mini column, and repeat step 6. If the sample volume was
greater than 140 μl, repeat this step until all the lysate has been loaded onto the spin
column.

8. Carefully open the QIAamp Mini column and add 500 μl Buffer AW1. Close the cap,
and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp Mini column in a
clean 2 ml collection tube (provided), and discard the tube containing the filtrate.

9. Carefully open the QIAamp Mini column and add 500 μl Buffer AW2. Close the cap
and centrifuge at full speed (20,000 x g; 14,000 rpm) for 3 min. Continue directly with
step 11, or to eliminate possible Buffer AW2 carryover, perform step 10 and then
continue with step 11.

11. Centrifuge at 6000 x g (8000 rpm) for 1 min.

A single elution with 60 μl Buffer AVE is enough to elute at least 90% of the viral RNA
from the QIAamp Mini column. Performing a double elution using 2 x 40 μl Buffer AVE
will increase yield by up to 10%. Elution with volumes of less than 30 μl will lead to
reduced yields and will not increase the final concentration of RNA in the eluate.
Viral RNA is stable for up to one year when stored at –30°C to –15°C or at –90°C to
–65°C.
Tetro cDNA Synthesis Kit Protocol

1. Vortex solutions and centrifuge briefly before use.

2. Prepare the priming mastermix on ice in an RNase-free reaction tube:

Component cDNA Control (-RT)

(+RT)

 Total RNA (up to 5μg) 5 μl 5 μl

or mRNA (up to 0.5μg)

 Primer: Oligo (dT)18 or

Random Hexamer (or 1 μl 1 μl
GSP) 1 μL
 5X RT Buffer 4 μl 4 μl
 10mM dNTP mix 1 μl 1 μl
 RiboSafe RNase
1 μl 1 μl
Inhibitor
 Tetro Reverse
Transcriptase (200 1 μl 0 μl
u/μL) ADD LAST
 DEPC-treated water / 7 μl / 8 μl
 TOTAL VOLUME: 20 μl

3. Mix gently by pipetting.

4. Incubate samples at 45 °C for 30 min. If using random hexamers,
incubate at 10 min at 25 °C followed by 45 °C for 30 min.
5. Terminate reaction by incubating at 85 °C for 5 min, chill on ice.
6. Store reaction at –20 °C for long term storage, or proceed to PCR
immediately.
This protocol is intended for use as a guide only; conditions will vary from
reaction to reaction and may need optimization.
Commented [Cl1]: 8 µL
Controls for RT-qPCR
A minus Reverse Transcription control (-RT control) should be included in all RT-qPCR experiments to test for contaminating DNA
(such as genomic DNA or PCR product from a previous run).
Such a control contains all the reaction components except for the reverse transcriptase. Reverse transcription should not occur in
this control, so if PCR amplification is seen, it is most likely derived from contaminating DNA.
 Photometry (UV-Vis) Fluorescence

How is the The photometric measurement of nucleic acids is based The fluorometric measurement of nucleic acids is based
optical on the intrinsic absorptivity properties of nucleic acids upon the use of fluorogenic dyes that bind selectively to
signal (DNA and RNA). When an absorption spectrum is DNA or RNA.
generated? measured, nucleic acids absorb light with a characteristic
peak at 260 nm.
 Fluorescent dyes selectively bind to DNA, RNA or protein. Dyes only emit
signal when bound to the target.

Typical RNA/DNA absorbance spectrum.

How is the  The signal is measured by spectrophotometers or  The signal is measured by fluorometers. Sample is
optical spectrometers. The attenuation in the light that excited with filtered light (at the excitation
signal
reaches the detector after passing through the sample wavelength, and the emitted light (at the emission
measured?
is measured in relation to the incident light and wavelength) is recorded by a detector.
expressed as absorbance values of the sample in the
solution.
  Wavelength separation can take place before or after  Wavelength separation can take place in various
the light has passed the sample, and the optical light ways (for example with filters or with
path can be horizontal or vertical. monochromators)

How is the  Concentrations of nucleic acids can be directly  Concentrations of nucleic acids are measured using
concentrati calculated from their measured absorbance values at the fluorescence signal of the sample and a
on of
nucleic 260 nm, using the Beer-Lambert's equation: calibration curve is generated from standard samples
acids of known concentration and fit to appropriate
calculated? regression models.

where:
o C= nucleic acid concentration in molar (M)
o A=UV absorbance in absorbance units (AU)
o ε=wavelength-dependent molar absorptivity
coefficient (or extinction coefficient) in M-1cm-1
o L= light path in cm (cm)
  This concentration calculation is automated in many
instruments.

Typical Fluorescence Standard Curve.

 The limit of detection and linear response of the
measurements are specific to each assay.

What are  It is simple—no sample preparation, dyes, or  It is specific—performed measurement is selective

the standards are required for DNA, ds DNA, ssDNA, and RNA
advantages
 Can provide direct measurements of purity ratios—  It is sensitive—can measure pg/mL; it is the
?
A260/280 and A260/230 recommended method for very diluted nucleic acid
 Can provide information on contaminants—can samples
identify non-nucleic acid contamination in samples  It is accurate despite contamination being present in
(proteins, phenol, guanidine salts) and provide the sample, including nucleic acid contaminants
 corrected concentrations (applicable to NanoDrop
One/OneC instruments)

What are  It is not selective—does not distinguish between  It takes longer—reagent and sample preparation are
the DNA or RNA required
disadvantag
 It has limited sensitivity—detection limits are higher  No purity information is provided
es?
than fluorescence-based methods

We offer a range of instruments to perform nucleic acid quantification using either photometry of fluorescence