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. 2022 Jan 20;23(1):66.
doi: 10.1186/s12864-021-08275-w.

Modified Northern blot protocol for easy detection of mRNAs in total RNA using radiolabeled probes

Tao Yang  1 , Mingdi Zhang  1 , Nianhui Zhang  2
Affiliations

Modified Northern blot protocol for easy detection of mRNAs in total RNA using radiolabeled probes

Tao Yang et al. BMC Genomics. .

Abstract

Background: Northern blotting is still used as a gold standard for validation of the data obtained from high-throughput whole transcriptome-based methods. However, its disadvantages of lower sensitivity, labor-intensive operation, and higher quality of RNA required limit its utilization in a routine molecular biology laboratory to monitor gene expression at RNA level. Therefore, it is necessary to optimize the traditional Northern protocol to make the technique more applicable for standard use.

Results: In this paper, we report modifications and tips used to improve the traditional Northern protocol for the detection of mRNAs in total RNA. To maximize the retention of specifically bound radiolabeled probes on the blot, posthybridization washes were performed under only with moderate-stringency until the level of radioactivity retained on the filter decreased to 20~50 counts per second, rather than normally under high and low stringency sequentially for scheduled time or under only high stringent condition. Successful detection of the low-expression gene using heterologous DNA probes in 20 µg of total RNA after a two-day exposure suggested an improvement in detection sensitivity. Quantitatively controlled posthybridization washes combined with an ethidium bromide-prestaining RNA procedure to directly visualize prestained RNA bands at any time during electrophoresis or immediately after electrophoresis, which made the progress of the Northern procedure to be monitored and evaluated step by step, thereby making the experiment reliable and controllable. We also report tips used in the modified Northern protocol, including the moderate concentration of formaldehyde in the gel, the accessory capillary setup, and the staining jar placed into an enamel square tray with a lid used for hybridization. Using our modified Northern protocol, eight rounds of rehybridization could be performed on a single blot. The modification made and tips used ensured the efficient proceeding of the experiment and the resulting good performance, but without using special reagents or equipment.

Conclusions: The modified Northern protocol improved detection sensitivity and made the experiment easy, less expensive, reliable, and controllable, and can be employed in a routine molecular biology laboratory to detect low-expressed mRNAs with heterologous DNA probes in total RNA.

Keywords: Detection sensitivity; Formaldehyde-agarose gel; Northern blot analysis; Posthybridization wash.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Effects of the modified Northern protocol on the performance of Northern blot analysis. a Germinating wheat was vernalized at 0~2 ºC for 0, 10, 20, and 30 d, respectively. The total RNA was isolated and an equal amount of total RNA (50 µg) was resolved using the formaldehyde-agarose gel containing 12% formaldehyde. Gel treatment, transfer of separated RNAs to a positively charged nylon membrane, fixation of transferred RNAs on the membrane, and prehybridization and hybridization were performed, as described in Methods. The traditional posthybridization washes under high and low stringency sequentially for scheduled time were performed according to Clark [20]. The probe used for detection was an Aox1 cDNA from N. tabacum. Hybridization with 18 S was used as an internal control. The exposure times for the detecting Aox1 and 18 S were 5 d and 30 min, respectively. b An equal amount of total RNA (20 µg) from vernalized germinating wheat at 0~2 ºC for 0, 10, 20, and 30 d, respectively, was loaded to analyze the level of Aox1 transcripts. Quantitatively controlled moderate-stringency washes were performed, as described in Materials and methods. The exposure times for the detection of Aox1 and 18 S were 2 d and 30 min, respectively. The values below the blot denote the fold-change relative to the germinating wheat without vernalization (0 d), standardized to the 18 S rRNA content. This experiment was performed twice with similar results. c An equal amount of total RNA (20 µg) from the leaves of a chlorophyll reduced mutant of B. napus (MT) and its wild type (WT) grown in the field at two-leaf stage was subjected to RNA gel blot analysis following the modified protocol, as described in Materials and methods. Eight different probes were used to rehybridize to the same blot. The experiment performed twice, and the quantification of the hybridization signals from the autoradiographs showed that there was no significant difference in the expression of these genes investigated between the mutant and the wildtype oilseed rape
Fig. 2
Fig. 2
Modified capillary transfer setup for blotting of size-separated RNA to a filter membrane. The transfer setup is the same as described in most protocols. Our modification was to add an accessory setup in accordance with Materials and methods to ensure good contact between each component of the setup, and no shift of the center of the gravity of the top weight as a result of the uneven pressure distribution of the top weight to the paper towels underneath
Fig. 3
Fig. 3
Gadgets used for hybridization. a Staining jar. Normally, the staining jar is used for staining tissue slices, where the gadget was used for prehybridization and hybridization, and its bottom size is 7.2 cm × 5.2 cm, with the thickness of its wall being 0.5 cm. The hybridization of the larger blotted membrane was performed in a glass Petri dish; b Enamel square tray with lid: During hybridization, the staining jar was placed into an enamel square tray, after being covered with the lid, the enamel tray was taken into a lab incubator to hybridize at 42 ºC. The thick glass of the staining jar, the metallic material and the enamel of the enamel square tray can protect investigators from hazardous radiation, and prevent the evaporation of toxic formamide and the spillage of radioactive materials
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