Processes 2018, 6,38
8. RNA
8.1. Literature Review
RNA is a major macromolecule class which contributes to ribosome assembly and cellular
information processing. Methods used for quantifying RNA include UV absorbance, orcinol
colorimetric reaction, and thiazole orange fluorescent dye [50]. UV absorbance is precise but requires
a pure sample and is not feasible for a mixture of DNA and RNA. Orcinol is not as precise for
RNA or for a mixture of DNA and RNA, and carbohydrates may also interfere. Thiazole orange
has good precision, but fluorescence is biased toward GC content, and it is less sensitive for RNA
than for DNA [50]. Additionally, kits are available for RNA extraction but focus predominantly on
downstream applications such as PCR and RNAseq, and thus remain questionable as quantitative
methods. Fluorescent dyes such as RiboGreen and PicoGreen have also been reported for quantifying
extracted RNA [52] but are usually used in combination with kit extractions.
Major concerns in selecting an RNA quantification method include sample purity, accuracy, and
bias of nucleotide content. Many studies have used the colorimetric orcinol reaction to quantify RNA
after hot perchloric acid extraction. However, Benthin et al. [67] developed an alternative method
with the bacterium Lactobacillus that utilizes alkali (KOH) lysis in combination with cold perchloric
acid extraction, followed by UV absorbance. The method provided similar accuracy to the orcinol
reaction but showed improved precision [67]. Benthin’s KOH-UV method was selected for the current
study as a more reliable and safe method, using cold rather than hot perchloric acid, and to eliminate
interference from carbohydrates, which occurs in the orcinol reaction. The results can be quantified
with UV absorbance using average nucleotide molar extinction coefficients, which eliminates the need
to prepare a standard from a different source. This method has been used in metabolic modeling
studies to quantify RNA percentage [68–70].
8.2. Procedure (After Benthin et al., 1991)
8.2.1. Reagents
•
Cell pellet (2–8 mg dry biomass, fresh or frozen, washed with carbon-free media).
•
HClO 4 solutions: 0.5 M, 0.7 M, and 3 M.
•
0.3 M KOH solution.
8.2.2. Assay
(1) Wash cell pellet three times with 3 mL 0.7 M HClO 4 to degrade cell walls. Vortex to re-suspend in
between washing. Centrifuge 4000 rpm for 10 min at 4 ◦ C and decant between washes.
(2) Re-suspend pellet in 3 mL 0.3 M KOH to lyse cells.
(3) Incubate in a 37 ◦ C water bath for 1 h, shaking at 15-min intervals.
(4) Cool and add 1 mL 3 M HClO 4 .
(5) Centrifuge and decant supernatant into a new 50-mL polypropylene centrifuge tube.
(6) Wash pellet twice with 4 mL 0.5 M HClO 4 (re-suspend and mix), centrifuge, and decant supernatant
into the 50-mL tube. The 0.5 M HClO 4 extracts the RNA, while DNA, which is stable even in strong
alkali, and protein, which does not solubilize in the alkali, remain in the precipitate.
(7) Add 3 mL 0.5 M HClO 4 to the collection of extracts to obtain a total volume of 15 mL,
and centrifuge once more to remove any non-visible precipitates of KClO 4 .
(8) Measure absorbance at 260 nm against a 0.5 M HClO 4 blank.
(9) Calculate RNA quantity by assuming 1 unit of absorbance at 260 nm corresponds to 38 µg/mL
RNA on average [71].
Notes: Quartz cuvettes are commonly used for measuring UV absorbance; however, disposable
UV cuvettes can also be used (VWR 47727-024, rated to 220 nm and tested for chemical compatibility
168
8. RNA
8.1. Literature Review
RNA is a major macromolecule class which contributes to ribosome assembly and cellular
information processing. Methods used for quantifying RNA include UV absorbance, orcinol
colorimetric reaction, and thiazole orange fluorescent dye [50]. UV absorbance is precise but requires
a pure sample and is not feasible for a mixture of DNA and RNA. Orcinol is not as precise for
RNA or for a mixture of DNA and RNA, and carbohydrates may also interfere. Thiazole orange
has good precision, but fluorescence is biased toward GC content, and it is less sensitive for RNA
than for DNA [50]. Additionally, kits are available for RNA extraction but focus predominantly on
downstream applications such as PCR and RNAseq, and thus remain questionable as quantitative
methods. Fluorescent dyes such as RiboGreen and PicoGreen have also been reported for quantifying
extracted RNA [52] but are usually used in combination with kit extractions.
Major concerns in selecting an RNA quantification method include sample purity, accuracy, and
bias of nucleotide content. Many studies have used the colorimetric orcinol reaction to quantify RNA
after hot perchloric acid extraction. However, Benthin et al. [67] developed an alternative method
with the bacterium Lactobacillus that utilizes alkali (KOH) lysis in combination with cold perchloric
acid extraction, followed by UV absorbance. The method provided similar accuracy to the orcinol
reaction but showed improved precision [67]. Benthin’s KOH-UV method was selected for the current
study as a more reliable and safe method, using cold rather than hot perchloric acid, and to eliminate
interference from carbohydrates, which occurs in the orcinol reaction. The results can be quantified
with UV absorbance using average nucleotide molar extinction coefficients, which eliminates the need
to prepare a standard from a different source. This method has been used in metabolic modeling
studies to quantify RNA percentage [68–70].
8.2. Procedure (After Benthin et al., 1991)
8.2.1. Reagents
•
Cell pellet (2–8 mg dry biomass, fresh or frozen, washed with carbon-free media).
•
HClO 4 solutions: 0.5 M, 0.7 M, and 3 M.
•
0.3 M KOH solution.
8.2.2. Assay
(1) Wash cell pellet three times with 3 mL 0.7 M HClO 4 to degrade cell walls. Vortex to re-suspend in
between washing. Centrifuge 4000 rpm for 10 min at 4 ◦ C and decant between washes.
(2) Re-suspend pellet in 3 mL 0.3 M KOH to lyse cells.
(3) Incubate in a 37 ◦ C water bath for 1 h, shaking at 15-min intervals.
(4) Cool and add 1 mL 3 M HClO 4 .
(5) Centrifuge and decant supernatant into a new 50-mL polypropylene centrifuge tube.
(6) Wash pellet twice with 4 mL 0.5 M HClO 4 (re-suspend and mix), centrifuge, and decant supernatant
into the 50-mL tube. The 0.5 M HClO 4 extracts the RNA, while DNA, which is stable even in strong
alkali, and protein, which does not solubilize in the alkali, remain in the precipitate.
(7) Add 3 mL 0.5 M HClO 4 to the collection of extracts to obtain a total volume of 15 mL,
and centrifuge once more to remove any non-visible precipitates of KClO 4 .
(8) Measure absorbance at 260 nm against a 0.5 M HClO 4 blank.
(9) Calculate RNA quantity by assuming 1 unit of absorbance at 260 nm corresponds to 38 µg/mL
RNA on average [71].
Notes: Quartz cuvettes are commonly used for measuring UV absorbance; however, disposable
UV cuvettes can also be used (VWR 47727-024, rated to 220 nm and tested for chemical compatibility
168