Modified Protocol for Isolation of High Quality RNA from the Matured Bark Tissue of tossa Jute

Abstract

Jute is classified as a bast fibre crop which is one of the most important natural fibre crops. Jute fibre is both economical and environmentally friendly and it can be utilized for minimizing the use of synthetic fibres. For the quality improvement of jute fibre, the molecular study of the genes linked with the fibre biogenesis is crucial. RNA isolation is a fundamental step for gene expression and transcriptomic studies of jute fibre. RNA isolation is restricted from jute especially from the well-developed bark tissues because it is rich in mucilage, polysaccharides, and phenolic compounds. In this study, a new method was optimized for extracting the total RNA from the field-grown bark tissues of two tossa jute varieties at 30 and 60 Days-After-Germination (DAG). Two-times use of CTAB extraction buffer with SDS and later addition of TRIZOL made the protocol simple, cost effective and minimal time consuming as compared to other methods. The A260/A280 value ranged between 1.99 ± 0.05 to 2.10 ± 0.02 which determined the purity of RNA. The range of the mean yield of the RNA was 232.38 ± 5.01 to 419.49 ± 9.43 µg/ gm of tissue which is abundant for further molecular analysis. For validation of the protocol, the RNA was converted into cDNA which was further amplified by semi quantitative-real-time-PCR using Cellulose synthase gene specific primers. The results indicated that the proposed RNA isolation method will be helpful in further downstream processing for the betterment of jute fibre quality.

Keywords:
Jute; Fibre; RNA isolation; cDNA; Ces

HIGHLIGHTS

The modified RNA isolation method from bark tissues was simple and cost effective.

The yield and quality of the RNA were good enough.

The isolated RNA can be utilized for gene expression studies.

INTRODUCTION

Jute, the “Golden fibre”, is the second most important fibre crops after cotton in the globe [¹]. The natural ligno-cellulosic bast fibre [²] of jute is not only environment friendly and biodegradable but also cost-effective. Jute belongs to the family Malvaceae having only two cultivated species, namely, Tossa jute: Corchorus olitorius L and white jute: Corchorus capsularis L [³,⁴]. The development of fibre cells is controlled genetically [⁵] and the jute fibre quality improvement requires the combination of the molecular and classical approaches of breeding. Isolation of good quality and sufficient quantity of Ribonucleic acid (RNA) from plant tissue is needed in the molecular studies like Complementary Deoxyribonucleic Acid (cDNA) sequencing (RNA-Seq) studies for identifying expressed transcripts, differential gene expression and allele specific gene expression studies [⁶-⁹].

RNA isolation from plant tissues is restricted by the presence of exogenous Ribonuclease (RNase), polysaccharides, polyphenols, mucilaginous compounds, and lignin [¹⁰] and is a major challenge, especially from the bast tissue of field grown jute [¹¹]. This is problematic from jute tissue because of the elevated amount of mucilage, polysaccharides and phenols [¹²,¹³] in comparison to other crop plants. Mucilage is a highly acidic and proteinaceous compound [¹⁴] which binds with secondary metabolites and further co-precipitate with RNA, reducing yield and quality of RNA [¹¹]. The concentration of the mucilage accentuate with the age of tissue, for this reason, it is even more problematic to remove mucilage during isolation of RNA from the developing bast fibre in the advance growth stages, especially in tossa jute [¹⁵].

Plant’s RNA is extracted either using any RNA isolation kit or using different Non-kit methods. Several methods were reported for plant RNA isolation from the polyphenols and polysaccharides rich Euphorbia tirucallii and Aloe barbadensis [¹⁶]; from secondary metabolites rich Rheum sp and Arnebia sp [¹⁷]; and from starch and polysaccharide rich cereals [¹⁸]. However, the protocol for RNA extraction from the field grown jute fibre at mature stages is very meagre. Extraction of RNA from 3 days old jute seedlings was carried out by Mahmood and coauthors, 2011 [¹³] which was not found effective at 65 Days After Sowing (DAS) bark tissue of jute by Choudhary and coauthors, 2016 [¹⁵]. Till now, only three methods were reported for isolating RNA from the stem or the bark tissue at different developmental stages where only one method [¹⁵] is used for isolating the RNA from 65 days old bark tissues. The other two methods extracted RNA from the 45 days old bast tissues [⁹] or stem [¹¹]. These three methods are time consuming, cumbersome and/ or limited by requiring expensive chemicals like Cesium chloride (CsCl₂). Cetyltri-methylammonium bromide (CTAB) method described by Doyle and Doyle (1987) [¹⁹], was also used for RNA isolation of jute by Ahmed and coauthors, 2019 [⁹]. A prepared extraction buffer and TRIZOL reagent were applied for RNA isolation from stored seeds of maize rich in starch by Dutta and coauthors, 2020 [²⁰]. The modified RNA isolation protocol described in this paper using both CTAB buffer and TRIZOL reagent successfully yielded good quality and sufficient quantity of RNA from the field grown jute bark tissue of two Tossa jute varieties at 30 DAG and 60 DAG.

Before RNA isolation, identification of the developmental stages with optimum enhancement of bundles of fibre cell was carried out by histochemical analysis and well-developed fibre bundles were observed from all of the samples. After isolation of RNA, RNA samples were run on agarose gel for evaluation, which showed clear bands of 28S rRNA, 18S rRNA and 5S rRNA and the concentration was measured by Microplate Spectrophotometer for checking the yield and quality of the RNA samples. The quality of the RNA samples was pure, the yields were adequate, where the average yield of the 60 DAG samples was higher than the reported average yield the 45 Days sample by Ahmed and coauthors, 2019 [⁹] and 65 DAS samples by Chowdhury and coauthors, 2016 [¹⁵]. The cDNAs were prepared from the RNA samples and then amplified by semi Quantitative real time PCR (semi qRTPCR) with Cellulose synthase (Ces) gene specific marker and this result validated the method. The objective of the present study was to establish a simple, less time consuming and cost-effective methodology to isolate good quality and quantity of total RNA from the matured bark tissue of tossa jute which is rich in mucilage, polysaccharides and phenols at their two developmental stages which are required for transcriptome and gene expression studies of jute.

MATERIAL AND METHODS

Plant materials

Two tossa jute varieties- JRO 524 and JBO 1 were used. JRO 524, having comparatively coarse fibre and JBO 1, having finer fibre were chosen based on the available report [²¹]. The seeds were sown during March-April, 2022 at Mondouri teaching Farm of Bidhan Chandra Krishi Viswavidyalaya (Latitude 22.94° N, longitude 88.52° E, altitude 9.75 MSL), West Bengal, India. Plant tissues were collected at two stages- 30 DAG and 60 DAG These two stages were selected because secondary phloic fibre separation from the cambium is essential for the development of bast fibre [²²] and previously, both primary phloic fibre (PPF) and secondary phloic fibre (SPF) were observed in normal tossa jute plant at 30 and 60 Days after sowing [²³].

Histochemical analysis

For the genetic regulation studies including fibre development and biosynthetic pathways associated with fibre, proper development of fibre cell bundles and its differentiation into PPF and SPF are required which were examined by histochemical analysis in the present study. Transverse sections of the lower stem of 30 DAG and 60 DAG old plants of the two varieties were taken and stained with safranin dye. The dye was washed with 95% ethanol and the slides were observed under a Zeiss Axioscope A1 microscope (ZEISS, Germany) and Axio Cam ERc5s camera system.

Steps for isolation of RNA from the bark tissue-

The CTAB based RNA isolation protocol was modified from Ahmed and coauthors, 2019 [⁹] and Dutta and coauthors, 2020 [²⁰]. All of the reagents were prepared in 0.1% DEPC treated water and autoclaved after that. All of the mortar-pestles, glass wares and plasticware were treated with 0.1% DEPC treated water and then autoclaved. The steps followed are:

1. 250mg fresh jute bark tissue was separated from the lower stem, collected in a pre-chilled mortar and pestle and cut into small pieces with sterilized scissor.

2. Liquid nitrogen was added to homogenize the tissue.

3. 1500µl CTAB extraction buffer [containing 100 mM Tris base, 20 mM Ethylenediamine tetraacetic acid (EDTA), 1.4 M sodium chloride (NaCl), 2% CTAB], 100µl 10% Sodium Dodecyl Sulfate (SDS), 20µl 4% Polyvinylpyrrolidone (PVP), 15µl 2-mercaptoethanol and 100µl 1.4M NaCl were added, the tissue was homogenized and taken in a 2ml Eppendorf tube.

4. The tube was incubated at 65ºC for 20 minutes.

5. It was centrifuged at 13000rpm for 10 minutes at 4ºC in a cooling centrifuge machine (HERMLEZ 233 MK-2, HERMLE LABORTECHNIK, Germany).

6. The supernatant was taken in a fresh 2ml Eppendorf tube, 200µl extraction buffer, 50µl 2% SDS, 8µl 4% PVP, 8µl 2-mercaptoethanol and 20µl 1.4M NaCl were added, mixed, and kept in ice for 10 minutes.

7. It was centrifuged at 13000rpm for 10 minutes at 4ºC.

8. The supernatant was taken in a 2ml fresh tube and equal volume of Phenol: Chloroform: Isoamyl alcohol (25:24:1) was added and mixed properly.

9. It was centrifuged at 13000rpm for 10 minutes at 4ºC.

10. The aqueous phase was taken in a fresh tube, equal volume of TRIZOL was added, mixed and kept at room temperature for 10 minutes.

11. Half volume of Chloroform: Isoamyl alcohol (24:1) was added and kept in ice for 10 minutes.

12. It was centrifuged at 13000rpm for 10 minutes at 4ºC.

13. The aqueous phase was pipetted out in a fresh 1.5ml tube, twice volume of pre chilled 2-Propanol and 1/10th volume of 1.4 M NaCl were added, mixed and the tube was kept at - 20ºC for 1 hour.

14. It was centrifuged at 12000rpm for 10 minutes at 4ºC.

15. The supernatant was discarded, in the tube 100 µl 70% ethanol was added and centrifuged at 8000rpm for 5 minutes at 4ºC. This step was repeated again.

16. The supernatant was discarded; again, it was centrifuged at 8000rpm for 5 minutes at 4ºC to remove the excess ethanol.

17. Residual ethanol was taken out with the help of micro-pipette without disturbing the pellet and the sample was dried in ice keeping the tube opened (~15 min) in Laminar air flow (KlenzFlo^TM, Klenzaids Contamination Controls Pvt. Ltd., Mumbai, India).

18. The pellet was dissolved in 25 µl DEPC treated water.

19. The sample was kept at -20°C for further use.

RNA evaluation

The RNA samples were run on 1.2 % agarose gel at 60 mV for checking the integrity. Microplate Spectrophotometer (BioTek EPOCH 2, BioTek Instruments, U.S.) machine was used for checking the purity and quantity of the RNA.

cDNA Preparation and Semi quantitative real time PCR analysis

The isolated RNA samples were treated with DNase I for removing any traces of genomic DNA. For making cDNA, 1µg of individual RNA samples was reverse transcribed using 1st strand cDNA synthesis kit (PrimeScript^TM 1st strand cDNA Synthesis Kit, Takara Bio, Japan). After making the cDNA, the cDNA was amplified by polymerase chain reaction in a thermocycler machine (Applied Biosystems, Thermo Fisher Scientific, USA) using Ces gene specific primers which was designed in IDT Primer Quest^TM Tool [²⁴] from the given gene ID (COLO4_23957) published by Islam and coauthors, 2017 [²⁵]. Sequence of the forward primer was 5′-CTCAGATCGTCTCAACCAAGTC-3′ and sequence of the reverse primer was 5′-TTTCCTCCCTTGAAGCCATAC-3′. The PCR products were run on 1.2% agarose gel with 100bp DNA ladder.

RESULTS

Identification of the bark developmental stage having optimum enhancement of fibre cell bundles using histochemical analysis

In this study, the lower stem of the two varieties was used for transverse sectioning at their 30 DAG and 60 DAG and stained with safranin dye. In all of the four tissues, fibre cell bundles were well developed and differentiated into PPF and SPF, but the fibre cell bundles of JRO 524 were prominent compared to the JBO1 at 30 DAG as well as at 60 DAG (Figure 1).

Isolation and checking of quality and quantification of RNA

In this protocol, high strength CTAB buffer along with PVP, SDS, NaCl and β-mercaptoethanol were used for cell lysis and two times CTAB buffer washes with other chemicals were carried out. Finally, the pellet was dissolved in 25µL of DEPC treated water and stored at -20 °C for further use. The integrity of the total RNA was checked on a 1.2 % agarose gel. Clear bands of 28S rRNA, 18S rRNA and 5S rRNA were observed from the 30 DAG and 60 DAG bark tissue samples of JRO 524 and JBO 1 (Figure 2). The range of the average RNA yields from the overall samples was 232.38 ± 5.01 to 419.49 ± 9.43 µg / gm of tissue as mentioned in Table 1 which was abundant, but the bark tissues at 30 DAG stage had more RNA yields than the bark tissues at 60 DAG.

Semi quantitative real time PCR analysis for RNA isolation protocol validation

Semi qRT-PCR analysis was used to validate the isolated RNA of bark tissues if it can be utilized for gene expression and other downstream applications. In this semi qPCR study, Ces gene specific primers were used. After the PCR amplifications, 99bp fragments were found in all of the four samples (Figure 3) indicating that this RNA isolation process can be used for studying the Ces gene expression.

Table 1
Quantification of the isolated RNA samples.

Variety name	Age (DAG)	Mean yield of RNA (µg/gm of tissue)	A260/280
JRO 524	30	367.79 ± 3.6*	2.04 ± 0.03
JBO 1	30	419.49 ± 9.43	2.10 ± 0.02
JRO 524	60	232.38 ± 5.01	1.99 ± 0.05
JBO 1	60	269.19 ± 2.38	2.09 ± 0.03

• ^*Data are presented as ± standard deviation with 3 replications.

Table 2
Comparison of this RNA isolation method with some previously published RNA isolation protocol of jute.

Methodology	Tissue	Required time	A260/A280	RNA Yield	Reference
Guanidine thiocyanate-ammonium thiocyanate - Sodium- chloride sodium citrate	7 days old seedling	~ 3 hours	NA	500 - 600 µg/ gram of tissue	[12]
Phenol/SDS method	3 days old seedling	~ 1 hour	1.88-1.90	8246.75- 8507.50 ng/g tissue	[13]
Hot borate buffer followed by isopycnic centrifugation (termed as HBIC)	7 DAG and 45 DAG stem	~ 6 hours	1.88 - 1.91	~ 350-500 µg /gm fresh tissue	[11]
Modified Phenol/SDS method	3 DAS seedling and 65 DAS field grown bark tissue	~ 4.8 hours	1.96± 0.011* (for seedling) and 1.85±0.020 (for 65 DAS bark)	353.7±8.1 (for seedling) 224.9±11.24 (for 65 DAS bark) µg /gm fresh tissue	[15]
CTAB method	Root, stem, bark, stick, flower, fruit and leaf tissues of 45 days plants	~ 6.1 hours	2.03-2.10	~ 68.32- 148.01 µg /gm fresh tissue	[9]
CTAB- TRIZOL method	30 DAG and 60 DAG bark tissues	~ 3.3 hours	1.99- 2.10	232.38 ± 5.01 - 419.49 ± 9.43 µg/ gm of tissue	This CTAB- TRIZOL method

• ^* Data are presented as ± standard deviation with 3 replications.

Figure 1
The transverse sections of lower stem stained with Safranin dye at 30 DAG samples of two jute varieties- JRO 524 and JBO 1 (Under 40X) and 60 DAG samples of two jute varieties- JRO 524 and JBO 1 (Under 20X). PPF- Primary phloic fibre bundle and SPF- secondary phloic fibre bundle.

Figure 2
RNA samples from two tossa jute (Corchorus olitorius) fibre at 30 DAG and 60 DAG stage which were separated on agarose gel.

Figure 3
Semi qRT PCR results of Ces gene where 100 bp DNA ladder was used.

DISCUSSION

For the genetics and genomics studies of fibre development and biosynthesis pathways linked with jute fibre quality, it is required to use the RNA samples of jute fibre at the stage when fibre cell bundles were developed properly and differentiated into PPF and SPF [¹⁵]. Earlier in a study of pathways linked with lignin biosynthesis of Corchorus capsularis fibre, bast tissues at 30 DAG and 60 DAG were used where qRT PCR analysis was also performed [²⁶]. A histochemical study of middle stem section of JRO 204 jute variety and its low lignin phloem fibre mutant with Maule reagent at 30 DAS showed formation of PPF and SPF in the wild type plant whereas in the mutant, only PPF was found [²³], though in this study, the formation of both PPF and SPF was observed in the lower stem of the two varieties. In this study, in the lower stems of 60 DAG plant, PPF and SPF were formed in both of the varieties which was found to be similar and correlated with the results of Choudhary and coauthors, 2016 [¹⁵] where the lower stem of 65 DAS- JRO 204 jute variety was stained with safranin. This result stated that the gene expression and transcriptome studies can be done at these two stages of jute.

There are few methodologies for RNA extraction published in recent years which were extracted from seedlings, root, stem, bark tissue at 45 DAG, 45 days old bark, stick, leaf, flower, fruit of jute plant [¹², ¹³, ¹¹, ⁹] but only one method was reported for isolating RNA from more developed bark tissue of jute at 65 DAS stage [¹⁵]. TRIZOL and RNA isolation kits were also used for RNA isolation of jute [²⁶]. TRIZOL, GITC and phenol/SDS methods were not found to be suitable for isolating RNA from 45 DAG stage jute stem which was observed by Samanta and coauthors, 2011 [¹¹], but their method took more time for centrifugation and expensive chemical like CsCl₂ was required. Good quality RNA could not be extracted using the protocols of Mahmood and coauthors, 2011 [¹³] and Samanta and coauthors, 2011 [¹¹] for 65 DAS old jute bark tissue, which was observed by Choudhary and coauthors, 2016 [¹⁵] because in the 60-65 days old jute bark tissue, mucilage, secondary metabolites, polysaccharides and phenols concentrations are very higher compared to seedlings. In the protocol of Choudhary and coauthors, 2016 [¹⁵], 3 hours incubation is required for precipitation of RNA which makes the protocol very tedious and it has also some complicated steps like two times pellet dissolving before dissolving it at the end with RNase free distilled water. In the process of RNA isolation from the stem and bark of 45 days jute plant developed by Ahmed and coauthors, 2019 [⁹], the RNA pellet was also dissolved twice before final dissolving which makes the procedure cumbersome and lengthy. Ahmed and coauthors, 2019 [⁹] used CTAB buffer for RNA isolation. Dutta and coauthors, 2020 [²⁰] observed that the use of RNA extraction buffer and TRIZOL were effective for isolation of good quality and quantity RNA from the starch rich stored seeds of maize and also time efficient, but it was found that two times buffer additions with CTAB and Polyvinylpyrrolidone (PVP) are required for removing mucilage and polyphenols of jute fibre which was not used in the method for seeds of maize. Based on the methods of Ahmed and coauthors, 2019 [⁹] and Dutta and coauthors, 2020 [²⁰], this new RNA isolation protocol from 30 DAG and 60 DAG bark tissue of field grown jute sample was developed.

Ahmed and coauthors, 2019 [⁹] stated that high strength CTAB lysis buffer with a high concentration of polyvinylpolypyrrolidone (PVPP), PVP and β- mercaptoethanol remove phenolic compounds, which oxidize and stick with RNA. They also found the effectiveness of two times buffer wash in lowering mucilage content, removing pigments and polyphenolic compounds. In jute fibre, protein contamination can be present due to the proteinaceous nature of mucilage which was removed by a strong detergent- SDS, as mentioned by Mahmood and coauthors, 2011 [¹³]. Application of EDTA as a chelator restricts the action of RNases and application of strong reducing agent like β-mercaptoethanol in the lysis buffer helps in disruption of di-sulphide bonds in RNases [²⁷]. Addition of Phenol: Chloroform: Isoamyl alcohol (25:24:1) helps to remove denatured proteins, pigments and assists in precipitating the huge quantity of polysaccharides which was seen in the bottom of the microcentrifuge tube. After Phenol: Chloroform: Isoamyl alcohol (25:24:1) addition, Chloroform: Isoamyl alcohol (24:1) addition was found to be more effective [²⁸]. TRIZOL is a popular reagent for RNA isolation, used for maintaining the integrity of RNA. Application of chloroform after TRIZOL addition followed by centrifugation helps in separating the solution in an aqueous phase where RNA is present and an organic phase where the DNA and proteins are present [²⁹]. In this method, addition of Chloroform: Isoamyl alcohol (24:1) after TRIZOL was used instead of only chloroform which was reported to be effective by Wei and coauthors, 2019 [³⁰]. RNA precipitation was carried out with isopropanol and NaCl. Application of NaCl minimizes the viscosity of mucilage [¹⁵] Washing the RNA pellet twice with 70% ethanol is essential for the purity of RNA [³¹].

Previously it was observed that the stem of jute at 7 DAG stage had more RNA yield than the jute stem at 45 DAG Stage [¹¹] and the RNA quantity at seedling stage was high than the RNA quantity of bark tissue of jute at 65 DAS stage [¹⁵] indicating the yield of the RNA reduces when the growth increases which was found for this method. The A260/A280 ratio ranges 1.99 ± 0.05 to 2.10 ± 0.02 (from Table 1) indicating the RNA samples are pure. The comparison between different published RNA isolation protocols of jute is listed in Table 2. Based on comparison with other protocols (Table 2), this method was found to take lesser time for isolation of RNA from the field grown bark tissue of older plants especially at 60 DAG stage which was validated by using two tossa jute varieties. The average yields of the 60 DAG bark tissues of both varieties were higher compared to the 45 Days different tissues of jute including bark as reported by Ahmed and coauthors, 2019 [⁹] and bast tissue at 65 DAS stage as reported by Choudhary and coauthors, 2016 [¹⁵].

Cellulose synthase (Ces) complex synthesizes cellulose which is a crucial component of secondary cell wall in the jute fibre, found around 60% in the fibre and provides the strength of the fibre [²²,³²]. In this study, semi qPCR analysis was performed using Ces gene specific primers. After the PCR amplifications, the 99bp single band from each of the samples after PCR amplification signified that that this RNA isolation process can be utilized for the Ces gene expression studies and others.

CONCLUSION

There are very limited protocols reported for extracting total RNA from the field grown mature bark tissue of jute because the tissue is rich in mucilage, polysaccharides and phenols which hinder and adversely effect the RNA isolation. The previously reported methods are time consuming, cumbersome, and sometimes costly chemicals are required. For these reasons, a new RNA isolation protocol from the bark tissue of two field grown tossa jute variety JRO 524 and JBO 1 at two developmental stages - 30 DAG and 60 DAG was derived using CTAB buffer and TRIZOL. The results showed that this RNA isolation protocol is not only simple and cost effective, but also it requires less time compared to other methods especially for well-developed field grown bark tissue. The isolated RNA samples can be called as pure based on the quality and the yields are also high and adequate for further studies like gene expressions. The qRT-PCR using Ces gene specific marker produced single 99 bp band for all of the samples which validate this methodology. This present protocol will be effective for the expression studies of different genes involved in jute fibre biogenesis and in the transcriptomic studies at different developmental stages which are required for genomics studies and genetic manipulations for fibre quality improvement of jute.

Acknowledgments

The authors are grateful to AINP on J&AF, BCKV and CRIJAF and the AICRP on medicinal and aromatic crops and betel vine projects for providing facilities to conduct the experiments.

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• Funding:

  This research received no external funding.

Edited by

Editor-in-Chief:

Bill Jorge Costa

Associate Editor:

Marcos Pileggi

Publication Dates

• Publication in this collection
  09 Aug 2024
• Date of issue
  2024

History

• Received
  26 June 2023
• Accepted
  08 Dec 2023