Estimation of Telomeric Repeat-containing RNA from DNA/RNA Hybrid Complexes

Summary

This protocol enables the isolation of DNA:RNA hybrids and their associated RNA (including TERRA) across multiple tissues. While our approach captures a subset of hybrid interactions, additional genomic mapping would be required for full genome-wide characterization.

Abstract

Telomeric repeat-containing RNA (TERRA) is a long non-coding RNA transcribed from all telomeres in higher eukaryotes, and only a subset of these molecules forms stable DNA/RNA hybrids at telomeres. We have outlined a detailed molecular protocol to identify and purify these hybrid estimates. This streamlined method enables the direct extraction of DNA/RNA hybrid interactions naturally formed in vivo and can be applied to detect all such regions. By minimizing procedural steps, this technique is highly efficient in isolating RNA attached to DNA, estimating the reliability and reproducibility of downstream sequencing. Guanidine isothiocyanate reagent is used for total cell lysis and homogenization, preserving the integrity of nucleic acids (DNA and RNA), particularly DNA/RNA hybrids. Adding chloroform initiates phase separation via centrifugation (three distinct layers): a lower phenol-chloroform phase, an interphase, and an upper aqueous phase. RNA, released from both the nucleus and cytoplasm, is retained in the aqueous phase, while high-molecular-weight DNA, including the DNA/RNA hybrid regions, is confined to the interphase. With isopropyl alcohol, followed by centrifugation, the aqueous phase (free RNA), interphase, and DNA, particularly DNA/RNA hybrids, are recovered. To remove the remaining traces of proteins, the sample is incubated with Proteinase K. Treatment with DNase, phenol/chloroform extraction, isopropyl alcohol, and centrifugation is used to release RNA hybridized to DNA.

Introduction

Transcription of the genome results in the formation of temporary hybrid molecules, known as R-loops, which consist of three-stranded structures. These structures include the non-coding and coding strands of DNA, with the non-coding strand displaced by the formation of hybrids with the complementary RNA¹. During genome transcription, a large number of non-coding RNAs are produced at varying rates from both DNA strands. Most of these RNAs serve as short-lived intermediates, but some play critical roles in the regulatory networks governing genome biology, particularly those originating from non-coding regions such as telomeres and centromeres

Protocol

All experimental protocols involving animals were approved by the Erciyes University Animal Ethics Committee (license number 19-07-2019, decision no. 19/127). Balb/c mice were used in this study: five healthy females, 6 weeks of age (25 g), and five males, 8 weeks of age (30 g). Mice were cared for and treated according to the Principles of Laboratory Animal Care (European rules). The study presented here utilized blood samples from 13 healthy control subjects, derived from a previous research project that received ethical approval from the Erciyes University Human Ethics Committee (Decision No: 17/002). A graphical abstract for this protocol is provided in <....

Results

In this study, we quantified and compared the levels of free TERRA, DNA/RNA hybrid TERRA, and telomere length across multiple mouse tissues (blood, sperm, skin, hypothalamus, pituitary, adrenal) as well as in human blood and skin samples, to investigate tissue-specific dynamics and the relationship to telomere biology. Free TERRA was isolated from the aqueous phase following phenol-chloroform extraction (protocol steps 3.6.1-3.6.5), enriching for cytoplasmic and nucleopla.......

Discussion

A central strength of the protocol presented here lies in its stepwise extraction and discrimination of free TERRA and DNA/RNA hybrid TERRA from complex tissue samples. Critical steps include the careful separation of the aqueous and interphase fractions during phenol-chloroform extraction, as cross-contamination can confound the distinction between free and hybrid TERRA. The subsequent DNase I treatment of the interphase fraction is also pivotal: insufficient digestion may leave DNA-bound RNA inaccessible, while overdig.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.2 mL PCR tubes	Greiner	671201
15 mL Falcon Tubes	Isolab	078.02.001
2 mL Syringe	Hayat	N/A
96- Multiwell plate	Greiner	50-720-3203
Absolute Ethanol	Merck	1070172511
Amonnium Acetate	Sigma	238074-500G
cDNA Synhesis Kit	Roche	07 912 439 001
Chloroform	Merck	1024451000
DNAse I Kit	Zymo	E1010
EDTA	Sigma	E9884-1KG
Glass bottle	Isolab	LB.IS.061.01.901
Graduated cylinder	Isolab	015.01.901
Guanidine isothiocyanate	Sigma	G9277-100G
Isoproponal	Merck	1096342511
Light Cycler 480 II	Roche	5015278001
Microcentrifuge Tubes (1.5 mL)	Greiner	616201
Micropipettes (10 µL)	Gilson	FD10001
Micropipettes (100 µL)	Gilson	FD10004
Micropipettes (1000 µL)	Gilson	FD10003
Micropipettes (2 µL)	Gilson	GFAM00064
Nanodrop	Shimadzu	C101-E112
PBS Tablet	Sigma	P4417-50TAB
PCR Device	Sensoquest	T3-0140
PGL3 Basic Vector	Promega	#212936
Pipette tips (10 µL, 200 µL, 1000 µL)	Greiner	772352
Plastic Petri dishes	Greiner	627102
Proteinase K	Sigma	P6556-10MG
SDS	Sigma	8170342500
Serological Pipette	Greiner	760107
Spin	Gilson	PMC880
Sybr Green Master	Roche	4707516001
Thermomixer	Eppendorf	#5355
Trizma base	Sigma	1503-1KG
Trizol	Biorad	7326890
Unimax 1010 DT Shaker	Heidolph	543-12310-00-6
Vortex	Heidolph	541-10000-00-1
Zymo-Spin I Columns	Zymo	C1003-50