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Glycogen

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Glycogen is a complex carbohydrate molecule that serves as the primary storage form of glucose in the body. It is found predominantly in the liver and skeletal muscles, where it can be readily converted into glucose to provide energy for cellular processes.

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Lab products found in correlation

Trizol reagent, by Thermo Fisher Scientific (13 mentions) Trizol, by Thermo Fisher Scientific (12 mentions) Proteinase k, by Thermo Fisher Scientific (7 mentions) Chloroform, by Merck Group (7 mentions) Nanodrop, by Thermo Fisher Scientific (5 mentions) Chloroform, by Thermo Fisher Scientific (4 mentions) Isopropanol, by Merck Group (4 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions) Hiseq 2500, by Illumina (3 mentions) Isopropanol, by Thermo Fisher Scientific (3 mentions) Truseq rna stranded sample kit, by Illumina (3 mentions) Mircury rna isolation kit biofluid, by Qiagen (2 mentions) Geneamp pcr kits, by PerkinElmer (2 mentions) Kapa hotstart readymix, by Roche (2 mentions) Viia7 real time pcr instrument, by Thermo Fisher Scientific (2 mentions) Proteinase k, by Roche (2 mentions) Dnase 1, by Roche (2 mentions) Trizol, by Roche (2 mentions) Bioanalyser 2100 system, by Agilent Technologies (2 mentions) Acna 3 m, by Merck Group (2 mentions)

Spelling variants of this product

RNase-free glycogen (7 citations) Pure glycogen (3 citations)

74 protocols using glycogen

1

Optimizing Extraction of Circulating RNA

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RNA fragments of less than 1000 nucleotides were isolated from 300 μl serum or plasma samples using the miRCURY RNA Isolation Kit—Biofluids (Exiqon, Vedbaek, Denmark). RNA isolation was performed according to the manufacturer’s instructions and included the use of recombinant DNAse. Twenty femtomolar of cel-miR-39-3p (Qiagen, Hilden, Germany) was spiked in during lysis of the sample. RNA was eluted in 30 μl of nuclease-free H2O and stored at −80 °C. To test the role of different RNA carriers, we isolated RNA under four different conditions: (1) no RNA carrier; (2) addition of 20 μg of glycogen (20 mg ml−1) (Roche); (3) addition of 1 μg of MS2 (0.8 μg μl−1) (Roche); and (4) joint addition of 20 μg of glycogen and 1 μg of MS2. Isolations were performed in triplicate.
Poel D., Buffart T.E., Oosterling-Jansen J., Verheul H.M, & Voortman J. (2018). Evaluation of several methodological challenges in circulating miRNA qPCR studies in patients with head and neck cancer. Experimental & Molecular Medicine, 50(3), e454-.
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2

Optimized RNA-seq Library Preparation

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We prepared the library following published protocols16 (link), 53 (link), using 40 μg K-562 total RNA plus 5.12 μl pooled capped ERCC spike-in RNA with the following modifications. (1) We used 0.5 μl glycogen (Roche) as the carrier instead of ethachinmate in the ethanol precipitation step. (2) We used KAPA HotStart Ready Mix (KAPA Biosystems) instead of GeneAmp PCR kits (PerkinElmer) for PCR amplification. (3) We selected 250 to 600 bp instead of 150 to 250 bp PCR products for sequencing.
Adiconis X., Haber A., Simmons S.K., Moonshine A.L., Ji Z., Busby M.A., Shi X., Jacques J., Lancaster M.A., Pan J.Q., Regev A, & Levin J.Z. (2018). Comprehensive comparative analysis of 5’ end RNA sequencing methods. Nature methods, 15(7), 505-511.
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3

Quantifying Viral RNA and Gene Expression

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Total RNA was extracted from cells using the guanidinium isothiocyanate extraction method [29 (link)] after adding 20 µg of glycogen (Roche) per sample as a carrier. The content of HCV RNA, ERLIN1 and ERLIN2 mRNAs and GAPDH mRNA (for normalization) in each sample was quantitated in a two-step RT-qPCR assays using the High-Capacity cDNA Reverse Transcription Kit and the Maxima SYBR Green/ROX qPCR Master Mix (2X) (Thermo Scientific). 10-fold serial dilution of plasmids containing each target sequence was used to prepare the standard curves that were used with each corresponding pair of primers: HCV (5′-TCTGCGGAACCGGTGAGTA-3′ and 5′-TCAGGCAGTACCACAAGG-3′); ERLIN1 (5′-GGGGTTGGTGGCTGTCCTGC-3′ and 5′-TAGCCTGGTCCACTGGGGCT-3′); ERLIN2 (5′-TGTGCACACGCTTCAAGAGGTCTA-3′ and 5′-AATGACCAGCCCAGGGGCCA-3′) and GAPDH (5′-GAAGGTGAAGGTCGGAGTC-3′ and 5′-GAAGATGGTGATGGGATTTC-3′). Results were normalized using GAPDH mRNA levels and were displayed in the figures as relative values compared to control cells.
Whitten-Bauer C., Chung J., Gómez-Moreno A., Gomollón-Zueco P., Huber M.D., Gerace L, & Garaigorta U. (2019). The Host Factor Erlin-1 is Required for Efficient Hepatitis C Virus Infection. Cells, 8(12), 1555.
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4

HPLC Purification of mRNA Variants

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Four of the mRNAs with different nucleotide chemistries were purified by HPLC using 7.8 × 50 mm alkylated non-porous polystyrene-divinylbenzene (PS-DVB)-based RNASep Prep RNA purification column (ADS Biotech, Hillington Park, Glasgow). WAVE Optimized Buffer A contained 0.1 M triethylammonium acetate in water (ADS Biotech), and WAVE Optimized Buffer B, composed of 0.1 M TEAA in 25% Acetonitrile (ADS Biotech), were used as the buffer system throughout. The purification was done according to the previously published protocol.31 (link) The collected fractions were desalted via Amicon Ultra-15 centrifugal filter unit (30 K membrane) (Merck Millipore), and the mRNA samples were subsequently recovered from fractions using overnight precipitation by 1:10 vol NaOAc and 1 vol isopropanol and glycogen (Roche).
Moradian H., Roch T., Anthofer L., Lendlein A, & Gossen M. (2022). Chemical modification of uridine modulates mRNA-mediated proinflammatory and antiviral response in primary human macrophages. Molecular Therapy. Nucleic Acids, 27, 854-869.
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5

Biotinylation of Spike-in RNAs

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Total RNA was spiked with in vitro transcribed 4sU-labeled FLuc and non-labeled RLuc RNAs and biotinylated using MTSEA biotin-XX (Biotium) as described by [18 (link)]. Briefly 80 μg total RNA was incubated with 8 ng FLuc and 4.8 ng RLuc (equimolar amounts, 130 amol), 10 mM HEPES pH 7.5, 1 mM EDTA and 5 μg MTSEA biotin-XX (freshly dissolved in DMF) in a total volume of 250 μl. Reactions were incubated 30 min in the dark at room temperature. Biotinylated RNA was recovered by extraction with one volume phenol: chloroform: isoamylalkohol (24:24:1) and separated using Phase-Lock-tubes (5Prime) by centrifugation (5 min, 20.800g, room temperature). RNA was precipitated by addition of 350 μl isopropanol, 25 μl 5 M sodium chloride and 1 μl glycogen (Roche Diagnostics, 20 μg/μl) to assist precipitation (30 min, 20.800g, 4°C). RNA was washed twice with 500 μl 80% ethanol in DEPC-H2O and dissolved in 25 μl DEPC-H2O (10 min, 55°C, shaking).
Uvarovskii A., Naarmann-de Vries I.S, & Dieterich C. (2019). On the optimal design of metabolic RNA labeling experiments. PLoS Computational Biology, 15(8), e1007252.
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6

Chromatin Immunoprecipitation with Flag-tagged Proteins

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1 ml of MNase-treated chromatin extract from above was incubated with 100 μl magnetic anti-Flag beads (prewashed 3 times in lysis buffer + 5 mg/ml BSA + 1.5 mM EDTA + cOmplete Mini protease inhibitor tablet Roche) overnight at 4 deg with rotation. Beads were washed 4 times with 1.5 ml lysis buffer + 1.5 mM EDTA and resuspended in 400 μl same buffer. DNA was eluted by the following: add 20 μg RNaseA and additional NaCl and EDTA so that the final concentrations increase by 100 mM (NaCl) and 10 mM (EDTA). Samples were incubated at 37 deg for 10 min with mixing. 0.5% SDS (final concentration) and 80 μg Proteinase K (Invitrogen) were added and samples incubated at 65 deg for 20 min with mixing. Supernatant was phenol/CHCl3/IAA extracted and DNA precipitated with 30 μg glycogen (Roche), 1/10 vol 3 M NaOAc, and 0.7 vol isopropanol. DNA was resuspended in 0.1X TE and quantitated by PicoGreen assay (Thermo Fisher).
Warfield L., Ramachandran S., Baptista T., Devys D., Tora L, & Hahn S. (2017). Transcription of nearly all yeast RNA Polymerase II-transcribed genes is dependent on transcription factor TFIID. Molecular cell, 68(1), 118-129.e5.
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7

Shearing and Purification of Genomic DNA

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Above plugs were washed 4 times for 15 minutes in 15 ml of TE buffer on a horizontal platform mixer at 180rpm at room temperature, transferred to 1.5ml eppendorf tubes, melted at 70°C for 2 minutes and equilibrated for 5 minutes in a water bath at 43°C. The plugs were then digested with 0.4U of GELase (Epicentre) for 45 minutes at 43°C and liberated DNA was cleaned by drop-dialysis (dialysis membranes 0.1µm VCWP04700 Millipore, MA, USA) against 15ml TE buffer for 1 hour. 0.1% of SDS was added to the DNA and treated with 80µg of proteinase K (Ambion) for 15 minutes at 50°C. This DNA was brought to a volume of 130 µl with TE buffer and transferred to a Covaris microTUBE AFA Fiber Pre-Slit Snap-Cap 6×16mm and sheared on a Covaris S220 sonicator for 4 min at 10% duty cycle, peak incident power 175, 200 cycles per burst in a water bath maintained at 4°C. Sonication under these conditions resulted in DNA fragments with a median shear length of 170bp. At this point, sheared DNA from the same sample in different plugs was combined. DNA was precipitated with 1µl of glycogen (Roche, 20 mg/ml) 0.1 volumes of 3M NaOAc pH5.2 and 2.5 volumes of 100% ethanol in dry ice for 15 minutes, and centrifuged at full speed in a standard microcentrifuge at 4 degrees for 15 minutes. The pellet was washed twice with 70% ethanol and solubilized in 70µl of TE low EDTA (10mM TrisHCl pH 8.0, 0.1mM EDTA).
Canela A., Maman Y., Jung S., Wong N., Callen E., Day A., Kieffer-Kwon K.R., Pekowska A., Zhang H., Rao S.S., Huang S.C., Mckinnon P.J., Aplan P.D., Pommier Y., Aiden E.L., Casellas R, & Nussenzweig A. (2017). Genome Organization Drives Chromosome Fragility. Cell, 170(3), 507-521.e18.
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8

Protein Expression and Purification

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Unless otherwise stated, oligonucleotides were from Eurofins Operon. KOD polymerase and Escherichia coli DE3 pLysS Rosetta cells were from Novogen. RNase inhibitor murine and T7 RNA polymerase were from New England Biolabs. Glycogen was from Roche, Proteinase K from Invitrogen, and Heparin Sepharose from Sigma.
The rapid DNA ligation kit was from Thermo Scientific. QIAquick PCR purification, QIAquick Gel elution, and QIAquick nucleotide removal kits were from Qiagen. The QuikChange site-directed mutagenesis kit was from Agilent. A 2 ml HiTrap heparin column and Superdex™ 200 gel filtration column were from GE Healthcare Life Sciences.
Singh M., Posse V., Peter B., Falkenberg M, & Gustafsson C.M. (2022). Ribonucleotides embedded in template DNA impair mitochondrial RNA polymerase progression. Nucleic Acids Research, 50(2), 989-999.
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9

Plasma miRNA Isolation Protocol

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Blood samples were processed within 24 h following withdrawal. To harvest cell-free plasma, samples were spun at 2,000 rcf for 5 min. Plasma was then transferred to a clean tube followed by centrifugation at 14,000 rcf for 10 min to remove cell debris. Samples were frozen at −80°C until further use. For HT-qPCR, miRNAs were isolated from 200 μL plasma with the miRNeasy Mini Kit (Qiagen, Hilden, Germany) using a modified protocol. In brief, 750 μL fresh QIAzol master mix (800 μL QIAzol and 1.25 μL 0.8 μg/mL MS2 RNA per sample) was added, followed by incubation at RT for 5 min. Following the addition of 200 μL chloroform, samples were incubated for 2 min at RT, followed by centrifugation for 15 min at 12,000 rcf at 4°C. Further preparation of the miRNA samples was performed according to the manufacturer's instructions. DNase digestion is generally not required since the combined QIAzol and RNeasy technologies efficiently remove most of the DNA without DNase treatment. Concentrations were measured with the Agilent 2100 Bioanalyzer using small RNA chips (Agilent, CA, USA). As human plasma samples contain only low amounts of extracellular cell-free RNA, glycogen (Roche, Germany) was applied as carrier RNA to enhance extraction efficiency and yields.
Kamhieh-Milz J., Moftah R.F., Bal G., Futschik M., Sterzer V., Khorramshahi O., Burow M., Thiel G., Stuke-Sontheimer A., Chaoui R., Kamhieh-Milz S, & Salama A. (2014). Differentially Expressed MicroRNAs in Maternal Plasma for the Noninvasive Prenatal Diagnosis of Down Syndrome (Trisomy 21). BioMed Research International, 2014, 402475.
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10

Selective Extraction and Detection of HBV cccDNA

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To selectively extract HBV cccDNA, infected HepG2-NTCP cells were lysed in 6-cm dishes with 1 mL of lysis buffer at 37°C for 60 min, and then, incubated with 0.25 mL of 2.5 M KCl overnight at 4°C. The lysis buffer contained 50 mM Tris–HCl (pH 7.4), 10 mM EDTA, 150 mM NaCl, and 1% SDS, without proteinase K. The lysate was clarified by centrifugation at 12,000 g for 30 min at 4°C. Viral DNA was extracted with phenol and phenol: chloroform, precipitated in an equal volume of isopropanol containing 20 µg glycogen (Roche), and dissolved in TE buffer. The prepared DNA sample was then treated with plasmid-safe adenosine triphosphate (ATP)-dependent deoxyribonuclease DNase (Epicentre Technologies) following the manufacturer’s instructions.
The treated Hirt DNA was subjected to Taq-man probe RT-qPCR for detecting the HBV cccDNA levels; the specific primers and the probe used are listed in the Supplementary (Table. S1).
Cai J., Li Y., Hu P., Xu R., Yuan H., Zhang W., Feng T., Liu R., Li W, & Zhu C. (2023). Plerixafor and resatorvid inhibit hepatitis B virus in vitro by upregulating elongation factor Tu GTP-binding domain containing 2. Frontiers in Cellular and Infection Microbiology, 13, 1118801.
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