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tRNA enrichment, quality control steps and expected outcomes of the Northern blot protocol (A) Bacterial total RNA and tRNA from Shewanella glacialimarina infected by bacteriophage (100 ng for each lane) analyzed on 10% urea-PAA gel to verify purity and quality of isolated tRNA. Note the enrichment of tRNA fragments (tRFs) in the tRNA fractions compared to total RNA. Numbers on the left correspond to the Low <t>Range</t> <t>ssRNA</t> Ladder. (B) Native 12% PAA gel analysis of 0.4 pmol of unlabeled (−) and labeled (+) probes confirming successful biotinylation. Ladder - Ultra-low range DNA ladder. Asterisks indicate biotin labeled probe carrying 1 (∗), 2 (∗∗) or 3 (∗∗∗) biotins. (C) Comparison of signal intensity between probes labeled with a single biotin or multiple biotins. (D) Analysis of bacterial total RNA (2 μg) by gel electrophoresis (left) and detection of tRNA-Leu and its stress-induced (phage infection) fragments (tRF; right) using long (14×10 cm) 12% urea-PAA gels. Ladder - Low Range ssRNA Ladder. (E) Detection of tRNA-Leu and tRNA-Leu fragment in Shewanella glacialimarina using a range (12.5–500 ng) of purified tRNA. tRNA sample is same as in panel A. (F) Typical Northern blot detection of bacterial tRNA-His. Total RNA (2 μg) and tRNA (200 ng) are separated on 10% urea-PAA mini (8.6×6.7 cm) gels (left), followed by verification of transfer (middle) and finally, tRNA-His is detected by chemiluminescent northern blot (right). Analysis is performed in duplicates (R1 and R2). (G) Utilization of chemiluminescent northern blot for detection of queuosinylated tRNA-His (right) in bacteria using 0.25% APB and TAE based urea-PAA mini gels (left). Δtgt serves as non-queuosinylated control of wild-type (wt) strain. Gel and samples were prepared as described in ref. (H) Chemiluminescent northern blot detection of thiolated tRNA-Glu (right) in yeast using 10% urea-PAA mini gels containing 10 μg/mL APM (left). Gel and transfer were performed as described in this protocol.
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tRNA enrichment, quality control steps and expected outcomes of the Northern blot protocol (A) Bacterial total RNA and tRNA from Shewanella glacialimarina infected by bacteriophage (100 ng for each lane) analyzed on 10% urea-PAA gel to verify purity and quality of isolated tRNA. Note the enrichment of tRNA fragments (tRFs) in the tRNA fractions compared to total RNA. Numbers on the left correspond to the Low <t>Range</t> <t>ssRNA</t> Ladder. (B) Native 12% PAA gel analysis of 0.4 pmol of unlabeled (−) and labeled (+) probes confirming successful biotinylation. Ladder - Ultra-low range DNA ladder. Asterisks indicate biotin labeled probe carrying 1 (∗), 2 (∗∗) or 3 (∗∗∗) biotins. (C) Comparison of signal intensity between probes labeled with a single biotin or multiple biotins. (D) Analysis of bacterial total RNA (2 μg) by gel electrophoresis (left) and detection of tRNA-Leu and its stress-induced (phage infection) fragments (tRF; right) using long (14×10 cm) 12% urea-PAA gels. Ladder - Low Range ssRNA Ladder. (E) Detection of tRNA-Leu and tRNA-Leu fragment in Shewanella glacialimarina using a range (12.5–500 ng) of purified tRNA. tRNA sample is same as in panel A. (F) Typical Northern blot detection of bacterial tRNA-His. Total RNA (2 μg) and tRNA (200 ng) are separated on 10% urea-PAA mini (8.6×6.7 cm) gels (left), followed by verification of transfer (middle) and finally, tRNA-His is detected by chemiluminescent northern blot (right). Analysis is performed in duplicates (R1 and R2). (G) Utilization of chemiluminescent northern blot for detection of queuosinylated tRNA-His (right) in bacteria using 0.25% APB and TAE based urea-PAA mini gels (left). Δtgt serves as non-queuosinylated control of wild-type (wt) strain. Gel and samples were prepared as described in ref. (H) Chemiluminescent northern blot detection of thiolated tRNA-Glu (right) in yeast using 10% urea-PAA mini gels containing 10 μg/mL APM (left). Gel and transfer were performed as described in this protocol.
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tRNA enrichment, quality control steps and expected outcomes of the Northern blot protocol (A) Bacterial total RNA and tRNA from Shewanella glacialimarina infected by bacteriophage (100 ng for each lane) analyzed on 10% urea-PAA gel to verify purity and quality of isolated tRNA. Note the enrichment of tRNA fragments (tRFs) in the tRNA fractions compared to total RNA. Numbers on the left correspond to the Low <t>Range</t> <t>ssRNA</t> Ladder. (B) Native 12% PAA gel analysis of 0.4 pmol of unlabeled (−) and labeled (+) probes confirming successful biotinylation. Ladder - Ultra-low range DNA ladder. Asterisks indicate biotin labeled probe carrying 1 (∗), 2 (∗∗) or 3 (∗∗∗) biotins. (C) Comparison of signal intensity between probes labeled with a single biotin or multiple biotins. (D) Analysis of bacterial total RNA (2 μg) by gel electrophoresis (left) and detection of tRNA-Leu and its stress-induced (phage infection) fragments (tRF; right) using long (14×10 cm) 12% urea-PAA gels. Ladder - Low Range ssRNA Ladder. (E) Detection of tRNA-Leu and tRNA-Leu fragment in Shewanella glacialimarina using a range (12.5–500 ng) of purified tRNA. tRNA sample is same as in panel A. (F) Typical Northern blot detection of bacterial tRNA-His. Total RNA (2 μg) and tRNA (200 ng) are separated on 10% urea-PAA mini (8.6×6.7 cm) gels (left), followed by verification of transfer (middle) and finally, tRNA-His is detected by chemiluminescent northern blot (right). Analysis is performed in duplicates (R1 and R2). (G) Utilization of chemiluminescent northern blot for detection of queuosinylated tRNA-His (right) in bacteria using 0.25% APB and TAE based urea-PAA mini gels (left). Δtgt serves as non-queuosinylated control of wild-type (wt) strain. Gel and samples were prepared as described in ref. (H) Chemiluminescent northern blot detection of thiolated tRNA-Glu (right) in yeast using 10% urea-PAA mini gels containing 10 μg/mL APM (left). Gel and transfer were performed as described in this protocol.
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tRNA enrichment, quality control steps and expected outcomes of the Northern blot protocol (A) Bacterial total RNA and tRNA from Shewanella glacialimarina infected by bacteriophage (100 ng for each lane) analyzed on 10% urea-PAA gel to verify purity and quality of isolated tRNA. Note the enrichment of tRNA fragments (tRFs) in the tRNA fractions compared to total RNA. Numbers on the left correspond to the Low <t>Range</t> <t>ssRNA</t> Ladder. (B) Native 12% PAA gel analysis of 0.4 pmol of unlabeled (−) and labeled (+) probes confirming successful biotinylation. Ladder - Ultra-low range DNA ladder. Asterisks indicate biotin labeled probe carrying 1 (∗), 2 (∗∗) or 3 (∗∗∗) biotins. (C) Comparison of signal intensity between probes labeled with a single biotin or multiple biotins. (D) Analysis of bacterial total RNA (2 μg) by gel electrophoresis (left) and detection of tRNA-Leu and its stress-induced (phage infection) fragments (tRF; right) using long (14×10 cm) 12% urea-PAA gels. Ladder - Low Range ssRNA Ladder. (E) Detection of tRNA-Leu and tRNA-Leu fragment in Shewanella glacialimarina using a range (12.5–500 ng) of purified tRNA. tRNA sample is same as in panel A. (F) Typical Northern blot detection of bacterial tRNA-His. Total RNA (2 μg) and tRNA (200 ng) are separated on 10% urea-PAA mini (8.6×6.7 cm) gels (left), followed by verification of transfer (middle) and finally, tRNA-His is detected by chemiluminescent northern blot (right). Analysis is performed in duplicates (R1 and R2). (G) Utilization of chemiluminescent northern blot for detection of queuosinylated tRNA-His (right) in bacteria using 0.25% APB and TAE based urea-PAA mini gels (left). Δtgt serves as non-queuosinylated control of wild-type (wt) strain. Gel and samples were prepared as described in ref. (H) Chemiluminescent northern blot detection of thiolated tRNA-Glu (right) in yeast using 10% urea-PAA mini gels containing 10 μg/mL APM (left). Gel and transfer were performed as described in this protocol.
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tRNA enrichment, quality control steps and expected outcomes of the Northern blot protocol (A) Bacterial total RNA and tRNA from Shewanella glacialimarina infected by bacteriophage (100 ng for each lane) analyzed on 10% urea-PAA gel to verify purity and quality of isolated tRNA. Note the enrichment of tRNA fragments (tRFs) in the tRNA fractions compared to total RNA. Numbers on the left correspond to the Low <t>Range</t> <t>ssRNA</t> Ladder. (B) Native 12% PAA gel analysis of 0.4 pmol of unlabeled (−) and labeled (+) probes confirming successful biotinylation. Ladder - Ultra-low range DNA ladder. Asterisks indicate biotin labeled probe carrying 1 (∗), 2 (∗∗) or 3 (∗∗∗) biotins. (C) Comparison of signal intensity between probes labeled with a single biotin or multiple biotins. (D) Analysis of bacterial total RNA (2 μg) by gel electrophoresis (left) and detection of tRNA-Leu and its stress-induced (phage infection) fragments (tRF; right) using long (14×10 cm) 12% urea-PAA gels. Ladder - Low Range ssRNA Ladder. (E) Detection of tRNA-Leu and tRNA-Leu fragment in Shewanella glacialimarina using a range (12.5–500 ng) of purified tRNA. tRNA sample is same as in panel A. (F) Typical Northern blot detection of bacterial tRNA-His. Total RNA (2 μg) and tRNA (200 ng) are separated on 10% urea-PAA mini (8.6×6.7 cm) gels (left), followed by verification of transfer (middle) and finally, tRNA-His is detected by chemiluminescent northern blot (right). Analysis is performed in duplicates (R1 and R2). (G) Utilization of chemiluminescent northern blot for detection of queuosinylated tRNA-His (right) in bacteria using 0.25% APB and TAE based urea-PAA mini gels (left). Δtgt serves as non-queuosinylated control of wild-type (wt) strain. Gel and samples were prepared as described in ref. (H) Chemiluminescent northern blot detection of thiolated tRNA-Glu (right) in yeast using 10% urea-PAA mini gels containing 10 μg/mL APM (left). Gel and transfer were performed as described in this protocol.
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tRNA enrichment, quality control steps and expected outcomes of the Northern blot protocol (A) Bacterial total RNA and tRNA from Shewanella glacialimarina infected by bacteriophage (100 ng for each lane) analyzed on 10% urea-PAA gel to verify purity and quality of isolated tRNA. Note the enrichment of tRNA fragments (tRFs) in the tRNA fractions compared to total RNA. Numbers on the left correspond to the Low Range ssRNA Ladder. (B) Native 12% PAA gel analysis of 0.4 pmol of unlabeled (−) and labeled (+) probes confirming successful biotinylation. Ladder - Ultra-low range DNA ladder. Asterisks indicate biotin labeled probe carrying 1 (∗), 2 (∗∗) or 3 (∗∗∗) biotins. (C) Comparison of signal intensity between probes labeled with a single biotin or multiple biotins. (D) Analysis of bacterial total RNA (2 μg) by gel electrophoresis (left) and detection of tRNA-Leu and its stress-induced (phage infection) fragments (tRF; right) using long (14×10 cm) 12% urea-PAA gels. Ladder - Low Range ssRNA Ladder. (E) Detection of tRNA-Leu and tRNA-Leu fragment in Shewanella glacialimarina using a range (12.5–500 ng) of purified tRNA. tRNA sample is same as in panel A. (F) Typical Northern blot detection of bacterial tRNA-His. Total RNA (2 μg) and tRNA (200 ng) are separated on 10% urea-PAA mini (8.6×6.7 cm) gels (left), followed by verification of transfer (middle) and finally, tRNA-His is detected by chemiluminescent northern blot (right). Analysis is performed in duplicates (R1 and R2). (G) Utilization of chemiluminescent northern blot for detection of queuosinylated tRNA-His (right) in bacteria using 0.25% APB and TAE based urea-PAA mini gels (left). Δtgt serves as non-queuosinylated control of wild-type (wt) strain. Gel and samples were prepared as described in ref. (H) Chemiluminescent northern blot detection of thiolated tRNA-Glu (right) in yeast using 10% urea-PAA mini gels containing 10 μg/mL APM (left). Gel and transfer were performed as described in this protocol.

Journal: STAR Protocols

Article Title: Protocol for rapid tRNA enrichment and chemiluminescent northern blot detection of tRNA and tRNA-derived fragments

doi: 10.1016/j.xpro.2026.104357

Figure Lengend Snippet: tRNA enrichment, quality control steps and expected outcomes of the Northern blot protocol (A) Bacterial total RNA and tRNA from Shewanella glacialimarina infected by bacteriophage (100 ng for each lane) analyzed on 10% urea-PAA gel to verify purity and quality of isolated tRNA. Note the enrichment of tRNA fragments (tRFs) in the tRNA fractions compared to total RNA. Numbers on the left correspond to the Low Range ssRNA Ladder. (B) Native 12% PAA gel analysis of 0.4 pmol of unlabeled (−) and labeled (+) probes confirming successful biotinylation. Ladder - Ultra-low range DNA ladder. Asterisks indicate biotin labeled probe carrying 1 (∗), 2 (∗∗) or 3 (∗∗∗) biotins. (C) Comparison of signal intensity between probes labeled with a single biotin or multiple biotins. (D) Analysis of bacterial total RNA (2 μg) by gel electrophoresis (left) and detection of tRNA-Leu and its stress-induced (phage infection) fragments (tRF; right) using long (14×10 cm) 12% urea-PAA gels. Ladder - Low Range ssRNA Ladder. (E) Detection of tRNA-Leu and tRNA-Leu fragment in Shewanella glacialimarina using a range (12.5–500 ng) of purified tRNA. tRNA sample is same as in panel A. (F) Typical Northern blot detection of bacterial tRNA-His. Total RNA (2 μg) and tRNA (200 ng) are separated on 10% urea-PAA mini (8.6×6.7 cm) gels (left), followed by verification of transfer (middle) and finally, tRNA-His is detected by chemiluminescent northern blot (right). Analysis is performed in duplicates (R1 and R2). (G) Utilization of chemiluminescent northern blot for detection of queuosinylated tRNA-His (right) in bacteria using 0.25% APB and TAE based urea-PAA mini gels (left). Δtgt serves as non-queuosinylated control of wild-type (wt) strain. Gel and samples were prepared as described in ref. (H) Chemiluminescent northern blot detection of thiolated tRNA-Glu (right) in yeast using 10% urea-PAA mini gels containing 10 μg/mL APM (left). Gel and transfer were performed as described in this protocol.

Article Snippet: Low Range ssRNA Ladder , New England Biolabs , N0364S.

Techniques: Control, Northern Blot, Infection, Isolation, Labeling, Comparison, Nucleic Acid Electrophoresis, Purification, Bacteria