Week 1: Total RNA Isolation, Quantification, and Integrity Assessment

Introduction

Obtaining pure and intact RNA is crucial for RNA manipulations, such as in a Northern blot, RT-PCR/RT-qPCR, RNA mapping, nuclease protection assays, in vitro translation, and cDNA library construction. Stabilizing RNAs in starting materials after sample collection and during isolation is crucial because changes in gene-expression patterns could occur due to specific and nonspecific RNA degradation and transcriptional inductions. Commercial RNA isolation kits, such as Qiagen RNeasy Plant Mini Kit,⁷ are available to isolate and purify total RNA from small quantities of plant material. Impurities, such as inhibitors, polysaccharides, and polyphenolics from the lysate, are removed before purification to increase RNA yield.⁸ Isolated RNA samples are treated with DNase to eliminate contaminating DNA. The digested mono- and oligonucleotides are removed later. A high-salt buffer is used to prevent small RNAs such as 5.8S rRNA, 5S rRNA, and tRNAs (account for 15–20% of total RNA) from binding to the silica-gel membrane.⁷ Thus, the purified RNAs are ready for RT-qPCR.

The standard protocol for plant RNA isolation is summarized in Figure 4-1. Leaf tissue is grounded in liquid N₂, followed by a strong denaturing buffer to inactivate endogenous RNases.⁷ After centrifugation, the supernatant is filtered through a filtration column to remove insoluble material and reduce the viscosity of the lysate. After further centrifuging, ethanol is added to the supernatant to provide a proper condition for nucleic acids, including RNA, to bind to the silica-gel-based column. After DNase treatment, the DNA contaminants are removed by washing them with a high-salt solution followed by a low-salt one. Finally, the bound RNAs are eluted with RNase-free water.

Efficient disruption and homogenization of the starting material is the prerequisite for total RNA isolation and purification.⁸ Complete disruption of cell walls, plasma membranes, and organelles is required to release the RNA in the sample. Different samples require different methods for complete disruption. Incomplete disruption results in significantly reduced RNA yields. Homogenization is necessary to reduce the viscosity of the lysates produced by disruption. Homogenization shears high-molecular-weight genomic DNA and other high-molecular-weight cellular components to create a homogeneous lysate. Incomplete homogenization results in inefficient binding of RNA to the RNeasy spin column membrane and, thus, significantly reduced RNA yields. Different disruption and homogenization methods are applied for different sample types. Some disruption methods also homogenize the sample, while others require an additional homogenization step.

The purity and quantity of RNA samples are determined by ultra violet (UV) absorbance and ratios using a UV spectrophotometer, such as a NanoDrop One spectrophotometer. For pure RNA, A260/A280 should be 1.8–2.2, and A260/A230 should be ~2.0.⁹ If A260/A230 is < 1.7–1.8, it suggests contamination. The A230 often remains constant for nucleic acid purified with a specific kit, while the amount of nucleic acid can vary due to the sample source.¹⁰ Thus, a low A260/A230 ratio can be due to a low yield, as often observed in RNA isolation. The disadvantages of this method are the lack of sensitivity to quantitate low-level nucleic acid samples and the fact that the degraded RNA samples still contribute to the 260 nm reading. Although the optical density (OD) ratios can be used to estimate nucleic acid purity, the presence of DNA, mono-nucleotides, and oligo-nucleotides in an RNA sample cannot be detected because they, too, absorb at 260 nm. Also, if significant amounts of contaminants with absorbance near 260 nm are present, they can contribute to the absorbance reading, thus overestimating nucleic acid quantity. For example, guanidine and other chaotropic salts or solvents absorb light at 230 nm, which can lead to higher 260 nm absorbance measurements through crossover.⁹

Instead of quantifying nucleic acids by directly measuring their UV absorbances, fluorescent dye-based quantification involves fluorescent binding dyes to dsDNA, RNA, and ssDNA, followed by measuring their fluorescence. One advantage of using fluorescent dye-based methods for RNA quantification is sensitivity. The NanoDrop One Spectrophotometer can detect as little as 2 ng/µl and up to 27,500 ng/μl (dsDNA) with extended auto-range pathlength technology. In contrast, dye-based methods, such as the NanoDrop 3300 Fluorspectrometer, are more sensitive than UV absorbance measurements. Its effective range of the assays can be increased to detect as little as 10 pg/µl but can only quantify up to 2.5 ng/µl DNA.¹⁰ Neither type of NanoDrop can provide information on nucleic acid integrity.

RNA is highly susceptible to degradation due to the ubiquitous presence of RNases. It is necessary to assess the integrity of RNA before downstream applications. Formaldehyde agarose/acrylamide gel electrophoresis and the use of a 2100 Bioanalyzer are two standard methods for assessing RNA quality.¹¹ In the former, the fluorescent dye, i.e., ethidium bromide, GelRed, or SYBR® Green, is incorporated into RNA in gels and visualized by the excitation of the dye under UV light. Instead of formaldehyde gel, the EMBER500™ RNA prestain is a more sensitive and convenient RNA detection dye, which can detect RNA on a regular agarose gel. For all the fluorescent dyes, RNA integrity is indicated by the staining intensity of the ribosomal RNA (rRNA) bands and degradation products. A 28S:18S rRNA ratio of 2:1 is considered good quality for mammalian rRNA.¹¹ The dye can also bind to genomic DNA, which can be identified due to its slower mobility than RNA in the gel. Although gel electrophoresis costs are low, it is labor-intensive, time-consuming, and requires up to µg of RNA to be visible in the gel. The RNA concentration can also be determined by comparing the relative fluorescence intensity of the RNA bands to those of known standards in the gel or by analyzing the gel image with a densitometer.

Although not being used in this lab module due to the high cost of reagents and chips, the 2100 Bioanalyzer is a chip-based capillary electrophoresis apparatus that analyzes nucleic acids and protein integrity with high resolution and automation.¹¹ Like DNA sequencing, most research labs outsource their RNA samples for integrity assessment. Samples are combined with a fluorescent dye, injected into wells on the chip, and electrophoresed through a gel matrix in the microchannels, followed by fluorescence detection. The data analysis software creates electropherograms and gel-like images for sizing and quantifying sample fragments. The assay only requires a small sample volume (1 µl), and data analysis takes ≤ 40 min. The results are derived from the entire RNA electrophoretic profile presented as a gel image, which shows an increase in the intensity of small RNA bands due to RNA degradation (Figure 4-2, left).¹¹ The image of the RNA ladder reveals the sample’s sizes and distribution of unknown RNAs by comparing them to those of an RNA standard with known concentrations. The results are also presented as the RNA Integrity Number (RIN), which ranges from 1 to 10, with 10 indicating the highest RNA integrity. As RNA degradation becomes obvious, peak heights for the 28S and 18S rRNA decrease, and smaller RNA peaks from degradation appear (Figure 4-2, right). The 28S and 18S peaks will be barely visible in significantly degraded RNA samples. It can also estimate RNA concentration by comparing peak areas of unknown samples. Unlike the NanoDrop spectrophotometer, the Bioanalyzer can’t directly measure RNA purity and requires more than one sample to determine DNA and protein contaminations.

For this lab exercise, total RNAs are isolated from Arabidopsis plants (leaves) with and without 96-hour dark treatment. The isolated RNA samples are then quantified and subjected to formaldehyde agarose gel electrophoresis to assess their integrity.

Procedure

The protocol for this lab module follows the procedure described in a previous paper by Chang et al.¹²

A. Total RNA Isolation with In-column DNase Treatment

Note: Odd-numbered groups: control plants without dark pretreatment. Even-numbered groups: dark-treated plants.

Perform steps 1–7 as precisely and quickly as possible.

1. Retrieve 1.5 ml microcentrifuge tubes, each containing ~100 mg leaf tissue from the -70°C freezer and place them in a container with liquid N₂.
2. Use a long forcep to retrieve a tube from the liquid N₂.
3. Quickly open the tube and push the leaf sample to the bottom with a blue plastic pestle.
4. Add 450 µl of the RLT lysis buffer and β-ME (10 µl per ml of RLT) into the tube before the samples thaw.
5. Grind the sample quickly and thoroughly with the plastic pestle for 30 sec without spilling.
6. Quickly vortex thoroughly.
7. Transfer the lysate to a QIAshredder spin column (purple) placed in a 2 ml collection tube.
8. Centrifuge at 13,000 rpm for 2 min.
9. Carefully remove the tube from the centrifuge and check if a pellet is at the bottom of the tube.
10. Use a 200 µl pipette to transfer the filtrate to a new 1.5 ml microcentrifuge tube and determine its volume. If there is a pellet, do not disturb it when taking out the filtrate. If you disturb the pellet, the purity will be low, and you won’t be able to quantify the sample correctly!
11. Add 0.5X the volume of 100% ethanol and mix by pipetting.
12. Transfer all the samples to an RNeasy spin column (pink) placed in a 2 ml collection tube.
13. Centrifuge at 13,000 rpm for 30 sec. Discard the flow-through, but keep the collection tube containing the nucleic acids.
14. Add 350 µl of Buffer RW1 and centrifuge at 13,000 rpm for 30 sec. Discard the flow-through.
15. DNase treatment:
    1. Pipette 80 µl of the DNase digestion mixture into the center of the binding column.
    2. Incubate at room temperature for 15 min.
16. Add 350 µl of Buffer RW1 and centrifuge at 13,000 rpm for 30 sec. Discard flow-through.
17. Add 500 µl of Buffer RPE and centrifuge at 13,000 rpm for 30 sec. Discard flow-through.
18. Add 500 µl of Buffer RPE and centrifuge at 13,000 rpm for 2 min. Discard flow-through.
19. Place the column into a clean 1.5 ml microcentrifuge tube; centrifuge at 13,000 rpm for 1 min.
20. Place the column into a new 1.5 ml tube without the cap (twist or cut it off).
21. Add 50 µl of RNase-free water to the center of the spin column and centrifuge at 13,000 rpm for 1 min to elute RNA.
22. Transfer the RNA into a new tube with a cap and store it in ice (Please keep all the nucleic acid samples on ice!)
23. Obtain your RNA sample’s O.D. ratios and concentration. Note: Remember to change Nanodrop’s “sample type” to RNA!

24. Store at -70°C for next week.

B. Assessing the integrity of the isolated total RNA with a formaldehyde agarose gel

Caution: Formaldehyde (F.A.) is toxic with known health effects, which include but are not limited to 1) irritation of the eyes, throat, nose, airways, and skin, 2) increased risk of cancer with long-term exposure to high levels, 3) lung injury, and increased asthma and allergy-related conditions, and 4) reproductive toxicity and allergic reactions from skin contact. Thus, steps 4 through 8 below should be performed in a fume hood, and all the waste produced should be treated as hazardous waste for proper disposal.

1. Weigh 0.6 g of agarose in a 250 ml flask
2. Add 2.5 ml of 20X F.A. gel buffer (see Equipment and Materials) and add 47.5 ml of RNase-free water.
3. Heat the mixture to melt agarose. Cool to 65°C in a water bath.

Note: The following steps need to be conducted in the fume hood.

4. Add 0.9 ml of 37% (12.3 M) formaldehyde (F.A.) and 5 µl of GelRed to the mixture and pour into a prepared gel tray.
5. After the gel solidifies, equilibrate it in sufficient 1X F.A. gel running buffer for ~30 min.
6. Add 1 volume of 5X RNA loading buffer (see composition in Equipment and Materials) to 4 volumes of RNA sample, ~4 µl of loading buffer and 16 µl of RNA, and mix.
7. For the RNA marker, one option is using 2 µl of RNA Millennium™ markers in 2 µl of 5X RNA loading buffer in 6 µl of RNase-free sterile water.
8. Incubate the RNA sample and markers for 3–5 min at 65°C, chill in ice for 2 min, and load all of each sample and markers in the 1X loading buffer into the well of the equilibrated F.A. gel.
9. Run the gel at 50 V in a 1X F.A. gel running buffer. Take a photograph of the gel.

Data Analysis and Discussion

1. After RNA isolation and purification, how do you know if your RNA sample is “pure” based on the results of O.D. ratios? Explain. (Hint: See Week 1 of Module 1.)
2. In addition to purity, the integrity of the RNA sample affects downstream manipulation.
   1. Can you use a regular agarose gel, like the one for DNA separation, to check RNA integrity? Why or why not? Explain.
   2. How do we evaluate the integrity of the isolated RNA sample? (Provide one method and briefly describe it.)
3. Why are 28S and 18S rRNA bands so bright, but you cannot see any band(s) for specific RNAs, such as RBCS1A and RBCS3B?