Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs

MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 μl of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression.     

Functional analysis of differential microRNA expression profile in mouse fibrotic liver tissues induced by CCl4

AIM:To discover the expression profile of microRNAs (miRNAs) in mouse fibrotic liver tissues induced by carbon tetrachloride (CCl4), and to investigate the functions of these differential miRNAs based on the gene ontology (GO) analysis and KEGG Pathway analysis. METHODS:The mice were randomly divided into normal group and model group. Liver fibrosis was induced by subcutaneous injection of CCl4. miRNA expression profile of the liver tissues was assayed by a mouse miRNA microarray (Agilent 12.0). The differential expression of miRNAs between the normal and model mice was screened, and GO analysis and KEGG Pathway analysis were performed to determine the functions of these differential miRNAs. RESULTS:Thirty-nine miRNAs with differential expression were discovered in the model mice compared with the normal mice, among which 23 were up-regulated and 16 were down-regulated. GO analysis and KEGG Pathway analysis indicated that most pathological processes of liver fibrosis regulated by miRNAs included cell proliferation and activation, cell apoptosis, cell cycle, cell adhesion, inflammatory reaction, cell migration, transforming growth factor β (TGF-β) signaling pathway, Wnt signaling pathway and proteometabolism process. GO analysis revealed that the key up-regulated miRNAs were mmu-miR-322, mmu-miR-15b, mmu-miR-195, mmu-miR-200b and mmu-miR-214, and the key down-regulated miRNAs were mmu-miR-16, mmu-miR-130a, mmu-miR-101b, mmu-miR-30a and mmu-miR-30e. Analyzing the target genes screened out by GO analysis and Pathway analysis simultaneously, we found that the key up-regulated miRNAs included mmu-miR-200b, mmu-miR-322, mmu-miR-106b, mmu-miR-23a and mmu-miR-15b, and the key down-regulated miRNAs included mmu-miR-16, mmu-miR-30e, mmu-miR-30c, mmu-miR-30a and mmu-miR-130a. CONCLUSION: Differential expression of miRNAs is discovered in mouse fibrotic liver tissues induced by CCl4 compared with the normal liver tissues. Most of the pathological processes involved in liver fibrosis may be regulated by miRNA, such as cell proliferation and activation, cell adhesion and apoptosis, cell migration and differentiation, metabolism, TGF-β receptor signaling pathway and so on.     

库尔勒香梨9个新miRNA克隆鉴定

利用在小RNA高通量测序试验中筛选出的脱萼组与宿萼组差异表达新miRNA基因,采用 Stem-loop法对总体表达量居前20位、在脱萼组和宿萼组中具有显著性差异表达、在子房和萼片组织中具有显著性差异表达的新miRNA进行成熟体的克隆鉴定、前体序列二级结构分析、qRT-PCR试验以及靶基因预测。结果显示,在不同的样本中有9个新miRNA（novel_miRNA）的成熟体序列以及4个novel_miRNA的表达量与高通量测序结果完全一致,并且预测得到大量具有生物学功能的靶基因。新发现的miRNA可能与‘库尔勒香梨’萼片脱落和宿存有密切关系。     

MicroRNA profile analysis of ischemic myocardial tissues from rats with acute myocardial infarction

AIM：To identify differentially expressed microRNAs (miRNAs) in ischemic myocardial tissues from the rats with acute myocardial infarction (AMI) by miRNA array technique, and to predict their targets and analyze their functions using bioinformatics. METHODS：The rat models of AMI (n=3) were prepared by ligaturing the left anterior descending coronary artery (LAD) of Wistar rats. Electrocardiogram and blood pressure were detected during the operation, and the myocardial infarct size was measured by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. Ischemic myocardial tissues were isolated from the infarct area 4 h after ischemia. The same procedure in sham group (n=3) was performed except for ligaturing LAD. Total RNA was extracted from ischemic and normal myocardial tissues. miRNA was isolated from total RNA, labeled with Cy3 and hybridized on miRNA array. Real-time PCR was applied to verify the reliability of miRNA array results. The targets of differentially expressed miRNAs were predicted and their functions were analyzed by bioinformatics. RESULTS：Rat model of AMI was successfully prepared and verified by electrocardiogram detection, blood pressure measurement and pathological observation. Compared with sham group, microarray screening showed that total 11 AMI-related miRNAs were selected, including 6 up-regulated and 5 down-regulated. Three of them (rno-miR-181c, rno-miR-146b and rno-miR-208) were related to the cardiovascular functions, while the functions of the others (rno-miR-672*, rno-miR-743b, rno-miR-128, rno-miR-138-1*, rno-miR-336, rno-miR-138-2*, rno-miR-325-3p and rno-miR-3572) were unknown and might be novel AMI-related biomarkers. Parts of the miRNA targets were also related to the cardiovascular functions. CONCLUSION：Differentially expressed miRNAs in AMI rats may serve as novel biomarkers for diagnosis of AMI and potential targets for treatment of AMI.     

Cloning and identification of novel miRNAs in the flower organs of Korla fragrant pear at anthesis

The objective of the study was to learn more about some newly identified miRNAs related to calyx persistence in Korla fragrant pear (Pyrus sinkiangensis Yu). Small RNAs were subjected to high-throughput sequencing after extraction from the ovaries and sepals of flowers with either a deciduous or a persistent calyx. Differentially expressed miRNAs were screened, and 73 new miRNAs were obtained. Twenty of these new miRNAs were selected to further validate all of the new miRNAs. Their mature miRNAs were cloned and identified, the secondary structures of the precursor miRNAs (pre-miRNAs) were analysed, and then qRT-PCR analysis was conducted. The results showed that the mature sequences of nine new miRNAs (novel_miRNA) in different samples were consistent with the results of high-throughput sequencing. Overall, this study improved current methods for the molecular cloning and identification of fruit tree miRNA. Nine new miRNA types were identified. This study laid a good foundation for elucidating the biological functions of these new miRNAs.     

Microarray-based miRNAs profiling in lower limb arteriosclerosis of diabetic rats

AIM: To establish the profiling of microRNAs (miRNAs) in the lower extremity arterial tissue between diabetic rats with lower limb arteriosclerosis (DAS) and diabetic rats with normal lower limb (DN), and to explore the possible molecular mechanisms involved in aberrant miRNA expression in DAS. METHODS: The rat models of DAS and DN were successfully established. The respective lower extremity arterial tissue was isolated. The total miRNAs were purified for a hybridization detection by miRNA microarray. The results of chip scanning and data were analyzed and verified by RT-qPCR. RESULTS: Ten miRNAs related to DAS, including rno-miR-206-3p, rno-miR-133a-5p, rno-miR-133b-3p, rno-miR-133a-3p, rno-miR-325-5p, rno-miR-675-3p, rno-miR-411-5p, rno-miR-329-3p, rno-miR-335 and rno-miR-126a-3p, were determined. All 10 abnormally expressed miRNAs were up-regulated. The validating results of RT-qPCR confirmed 9 of the miRNAs in line with chip expression. Just rno-miR-335 showed the opposite between PCR detection and microarray result. CONCLUSION: A group of miRNAs in diabetic rats suffering from lower limb arteriosclerosis plays an important role in the vascular atherosclerosis process. The abnormal expression of miRNAs is likely to affect the vascular atherosclerosis process.     

Differential microRNA expression profile in laryngeal cancer and effect of miR-125a-5p on proliferation of laryngeal cancer cell line

AIM：To investigate the differential microRNA expression profiles between laryngeal cancer and adjacent normal laryngeal mucosa. METHODS：Forty two pairs of laryngeal cancer tissue and adjacent normal laryngeal mucosa tissue were collected. Ten pairs of samples were used for determining microRNA expression by the method of miRNA microarray chip. Data analysis was performed to find out the significant differential microRNA expression profile in laryngeal cancer, and the difference was verified by quantitative real-time PCR (qRT-PCR) analysis on another 32 pairs of samples. Methyl thiazolyl tetrazolium (MTT) assay and colony-forming assay were used to analyze the proliferation of Hep2 cells induced by miR-125a-5p. RESULTS：Both miRNA microarray and qRT-PCR showed that the expression of let-7f-5p, miR-10a-5p, miR-125a-5p, miR-144-3p, miR-195-5p and miR-203 was down-regulated in laryngeal cancer tissues. miR-125a-5p suppressed the proliferation of Hep2 cells. CONCLUSION：The results of microarray are accordant with those of qRT-PCR. Significant difference of miRNA expression profiles between laryngeal cancer and adjacent normal laryngeal mucosa indicates that miRNAs may play a role in carcinogenesis and progression of laryngeal cancer. miR-125a-5p inhibits the proliferation of Hep2 cell, indicating a novel therapeutic target against laryngeal cancer.     

Effect of Epstein-Barr virus latent membrane protein 1 on oncomiRs expression profile in nasopharyngeal carcinoma cell line CNE1

AIM: To investigate the differential expression profile between nasopharyngeal carcinoma cell line CNE1 and its steady EBV-LMP1-transfected cell line CNE1-LMP1, and to explore the regulatory effect of LMP1 on oncomiRs expression in CNE1 cell line. METHODS: A microRNA array that targets 132 of the most well studied oncomiRs was used to detect the expression profile of CNE1 and CNE1-LMP1. qRT-PCR assay were used to verify the expression data detected by microarray. RESULTS: Among the restricted 132 miRNAs, 30 were detectable. Among which, 30 were expressed in CNE1-LMP1, 19 in CNE1 and 11 were specifically expressed in CNE1-LMP1. Among the 19 shared miRNAs, the expression level of 6 miRNAs (hsa-miR-19b, hsa-miR-17-3p, hsa-miR-22, hsa-miR-149, hsa-miR-150 and hsa-miR-188) elevated over two folds in CNE1-LMP1. No decrease in miRNA expression more than two folds was observed. qRT-PCR confirmed the expression difference of these six miRNAs (P<0.01). Among the 11 specifically expressed miRNAs in CNE1-LMP1, hsa-miR-122a showed the highest expression level surpassing the internal control sample. CONCLUSION: Our data suggest that LMP1 may play an important role in regulating the expression of miRNAs in tumor, which may be another important pathway employed by LMP1 in the development of nasopharyngeal carcinoma.     

枣树阶段转变相关microRNA家族的鉴定及其表达分析

与其他果树相比,枣树具有童期短、成花快的特征.已有研究表明,多个microRNA(miRNA)家族参与植物阶段转变和开花时间调控等过程.研究枣树阶段转变相关的miRNA家族对果树童期调控具有重要意义.以枣实生后代植株不同发育阶段(节位)的当年生枝(枣吊)为材料,通过Small RNA测序,在童期、过渡期和成年期等3个时...     

Effect of miRNAs on apoptotic leukemia cells induced by demethylcantharidin

AIM: To explore the role of miRNA expression in the process of antitumor drug demethylcantharidin (DMC)-induced leukemia cell apoptosis. METHODS: K562 cells were treated with DMC to induce apoptosis. Microarray assessment was performed to detect the changes of miRNAs. The expression of miRNAs was further detected by RT-PCR and real-time PCR. The miRNA-relative genes were analyzed by gene information softwares,and their expression was determined by ELISA analysis. RESULTS: The results of microarray showed that more than 290 miRNAs were differentially expressed after DMC treatment. The expression of miR-16, miR-34 and miR-125 increased at more than 1.5 folds in DMC treatment group determined by RT-PCR and real-time PCR analysis, while both miR-106 and miR-150 were down-expressed over 60％. Using microRNA TargetScan and miRanda analysis software, we found that the expression of oncogenes ( bcl-2, E2F1, E2F3 ) and tumor suppressor genes ( RB1, p53 ) may be regulated by the above miRNAs. The expression of RB1 and P53 proteins significantly increased, while Bcl-2, E2F1 and E2F3 proteins were obviously down-regulated after DMC treatment.CONCLUSION: DMC induces K562 cell apoptosis by regulating the expression of miRNAs and their relative genes.     

Effect of Ad-14-3-3σ on microRNA expression in different radioresistant nasopharyngeal carcinoma cells

AIM: To discuss the effect of Ad-14-3-3σ to microRNA (miRNA) in different radioresistant nasopharyngeal carcinoma (NPC) cells, CNE-1 and CNE-2, and study the relationship between the discrepancy of miRNA and radiosensitivity of NPC.METHODS: Ad-14-3-3σ was transfected to CNE-1 and CNE-2 cells, and then miRNAs were detected by Paraflo microfluidic microRNA chip. Hybridization images were collected using a laser scanner and the signals were normalized using a LOWESS filter. The effect of Ad-14-3-3σ to miRNAs and the relationship between the discrepancy of miRNA and radiosensitivity of NPC were studied according to Targetscan3.1 database (http://www.targetsan.org) after analyzing data.RESULTS: After treated by Ad-14-3-3σ, comparing to CNE-2 cells, there are 37 miRNAs changed remarkably, including 17 over-expression microRNAs and 20 under-expression microRNAs in CNE-1 cells. 6 miRNAs that one detective value was more than 1 000 and 3 folds than the other were hsa-miR-152,hsa-miR-205,hsa-miR-203,hsa-miR-7,hsa-miR-636 and hsa-miR-100.CONCLUSION: Ad-14-3-3σ can change the expression of miRNAs in different radioresistant nasopharyngeal carcinoma, and some miRNAs have relevance to carcinoma and radiosensitivity.     

Phosphorus toxicity in the Proteaceae: A problem in post-agricultural lands

South African Proteaceae are adapted to the low soil phosphorus (P) concentrations of the Cape Floristic Region. The efficient P uptake by Proteaceae means that these plants experience phosphorus (P) toxicity at lower rhizosphere [P] than crop plants. This is only problematic when cultivating Proteaceae (and many plants from this region) on previously agricultural land with high residual soil [P]. In this study we hypothesize that P toxicity will result in element imbalances in leaves of Proteaceae and information from this study aims to facilitate ameliorative treatments. Phosphorus toxicity was induced on-farm in Leucadendron ‘Safari Sunset’ (Proteaceae) with subsequent mapping of element distribution in non-necrotic leaf tissue using micro particle-induced X-ray emission spectrometry. Phosphate supply up to 0.01 mM in a fertigation solution resulted in increased stem length of Leucadendron ‘Safari Sunset’ while P concentrations in excess of this resulted in decreased stem length, increased leaf [P] up to 0.25% (w/w) and, between 1 mM and 5 mM P supply, typical P toxicity symptoms were observed. High P supply (5 mM P) resulted in increased leaf [P] in most leaf tissues including the epidermis, where calculations from an equilibrium speciation model indicated that there was 30% more dissolved PO₄³⁻ in the epidermis compared to leaves at low P supply (0 mM added P on soil with 34 mg P kg⁻¹). Concomitantly, bundle sheath and epidermal [Ca] were reduced and 10% more Ca was predicted to be adsorbed and precipitated as hydrapatite at high P supply. High P supply resulted in increased leaf [Cl] and [Mn] in all tissues studied; decreased total leaf [Fe], bundle sheath, xylem, phloem and epidermal [Fe] and decreased total leaf [Zn] and xylem and phloem [Zn]. The observed symptoms of P toxicity in Leucadendron ‘Safari Sunset’ (necrosis in some plants, chlorosis and leaf rosetting) co-occurred with (1) excess PO₄³⁻, which may bind Ca in the epidermis (leading eventually to necrosis); (2) reduced [Fe] and increased [Mn] (leading to chlorosis) and (3) reduced total and vascular [Zn] (leading to leaf rosetting).     

32种果树microRNA的生物信息学预测与分析

通过生物信息学预测miRNA的方法,采用基于生物信息学的基因搜索和同源搜索的方法成功地从NCBI数据库中登录的32种果树的EST中寻找miRNAs。从32种果树的774 400条ESTs中找到了110条miRNA前体序列,编码116条成熟体序列。利用miRbase数据库进一步分析,结果表明116个miRNAs属于45个miRNA家族,其中有7个保守的miRNA家族中的miRNA个数在5以上,20 个 miRNA家族的miRNA个数在 2 ~ 4,有18个miRNA家族的miRNA数量为1个。在柑橘、苹果、香蕉、猕猴桃和桃等果树中分别发现28、16、13、11和10个miRNAs。果树中miRNA的序列比较发现,相同家族的miRNA的序列存在一定水平的差异,其中碱基的变化包括相似频率的颠换与转换。     

Effect of the culture environment on stomatal features, epidermal cells and water loss of micropropagated Annona glabra L. plants

Shoots of Annona glabra L. were rooted in vitro under three levels of irradiance and two closure systems (conventional and natural ventilation) of the culture vessels. Once the shoots had been rooted, we studied how the manipulation of the culture environment affects the stomata features and water loss through leaf tissues after the plants are removed from the vessel. The stomata frequency increased significantly in the leaves of plants grown under high (300 μmol m⁻² s⁻¹) compared to low (50 μmol m⁻² s⁻¹) or intermediate (150 μmol m⁻² s⁻¹) irradiance, with higher effect under natural ventilation. Irrespective of the culture environment, leaves developed in vitro attained a higher stomata frequency than those grown in vivo. Under high irradiance and natural ventilation, the leaves presented functional stomata of characteristically elliptical shape and the epidermal cells were smaller and had slightly sinuous anticlinal walls. Besides, water loss through leaves of plants grown under high irradiance and natural ventilation was drastically reduced if these plants were exposed to an environment with low relative humidity thereafter. Our results indicate that an increased light availability and the use of natural ventilation improve the regulatory capacity of water loss in micropropagated A. glabra L. plants and can favor the plants’ survival and growth after transference to the natural environment.     

MicroRNA expression in hepatocytes with hydrogen peroxide-induced oxidative stress-injury and alleviating effect of mesenchymal stem cell-conditioned medium

AIM: To evaluate the changes of microRNA (miRNA) in hepatocytes during hydrogen peroxide-induced oxidative stress injury, and to observe the alleviating effect of mesenchymal stem cell-conditioned medium (MSC-CM) in this progress. METHODS: The hepatocyte oxidative stress injury model was established using hydrogen peroxide and human normal liver cell line L02. MSC-CM was prepared using centrifugation and filter. The effects of MSC-CM on hepatocyte injury were evaluated by apoptosis analysis, cell viability detection, cell cycle, and mitochondrial membrane potential (MMP). Twenty-one differentially expressed miRNAs were selected by gene chip hybridization, in which miR-143, miR-145, miR-301a and let-7a were confirmed by RT-qPCR. Bioinformatics software was utilized to predict target proteins of these miRNAs, and then the proteins were verified by Western blot.RESULTS: MSC-CM markedly attenuated hydrogen peroxide-induced oxidative stress injury by reducing apoptosis, promoting cell viability and regulating cell cycle. The expression of miR-143, miR-145, miR-301a and let-7a, indentified by RT-qPCR, increased under the condition of oxidative stress injury, while decreased after MSC-CM treatment. The expression of miR-143 predicted target proteins, HK2 and ADRB1, decreased under the hydrogen peroxide-exposure, while increased after MSC-CM treatment, which is consistent with the regulatory trend of miR-143. CONCLUSION: MSC-CM might attenuate hydrogen peroxide induced oxidative stress injury via inhibiting apoptosis and regulating some miRNA expression.     

嫁接对柑橘microRNA表达的影响

以‘锦橙’、‘资阳香橙’、‘飞龙枳’实生苗和‘锦橙’/‘资阳香橙’、‘锦橙’/‘飞龙枳’嫁接苗为试材,通过荧光定量PCR检测miRNA及其靶基因在嫁接苗和实生苗叶片和根系中的表达差异,分析嫁接对柑橘microRNAs及其靶基因表达的影响。结果证明嫁接对柑橘miRNA的表达有直接的影响。嫁接的影响更多地是促进接穗中一些与调控植物生长发育、胁迫应答及激素信号转导相关的miRNA的表达,并抑制了其对应靶基因的表达;而在砧木中,嫁接对根系的影响较多表现为抑制与植物生长发育、胁迫应答相关的miRNA的表达,促进其靶基因的表达。受影响的miRNA的种类及其表达的差异水平在不同砧木间有明显差异。     

Expression of miRNA-198 and miRNA-296-5P in PB3A-treated colon cancer cell line and its function prediction

AIM:To explore the effect of pinobanksin-3-acetate (PB3A) on microRNA (miRNA) expression profile of human colon cancer cells for providing new methods of treatment of colon cancer and development of targeted drug.METHODS:The method of miRNA expression profiling was used to observe the miRNA differential expression in human colon cancer SW480 cells after treated with PB3A.The expression of miRNA-198 and miRNA-296-5p in the SW480 cells was detected by RT-qPCR.The network databases of miRWalk,MicroT,miRanda and so on were used to predict the target genes regulated by these miRNAs,and pathway significant enrichment analysis was performed.RESULTS:miRNA microarray analysis showed that after treated with propolis flavonoid PB3A for 24 h,267 miRNAs with differential expression twice or more in the SW480 cells were observed.Among them,there were 30 miRNAs with 10-fold or more differential expression,in which 28 were up-regulated and 2 were down-regulated.The results of RT-qPCR showed that the expression levels of miRNA-198 and miRNA-296-5p were consistent with the results of miRNA microarray analysis,and the difference was statistically significant (P<0.05).Bioinformatic analysis revealed that miRNA-198 has 859 target genes,and miRNA-296-5p has 906 target genes.The target genes of miRNA-198 were clustered in pathways in cancer,axon guidance,Wnt signaling pathway,regulation of actin cytoskeleton,insulin signaling pathway and MAPK signaling pathway,while the target genes of miRNA-296-5p were clustered in axon guidance,Wnt signaling pathway,MAPK signaling pathway,endocytosis,melanogenesis,insulin signaling pathway and calcium signaling pathway.CONCLUSION:Propolis flavonoid PB3A affects the expression of miRNA in colon cancer SW480 cells.The abnormal expression of miRNA-198 and miRNA-296-5p may be involved in the inhibitory effect of PB3A on colon cancer.     

Up-regulation of microRNA-187* inhibits proliferation of human colon cancer cell line HCT116

AIM:To investigate the expression of microRNA-187* (miR-187*) in human colon cancer cell lines and normal colon tissues, and to determine the effects of miR-187* up-regulation on the proliferation and cell cycle of human colon cancer cell line HCT116. METHODS:The expression profiling of miRNAs in 3 colorectal adenocarcinoma samples and their matched normal tissue samples was performed using miRNA microarray chip. Total RNA was isolated from 8 colon cancer cell lines and 10 normal colon tissues. The miR-187* level was detected by Taqman real-time RT-PCR. B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1), the possible target of miR-187*, was also detected. Synthetic miR-187* mimics were transfected into HCT116 cell line by LipofectamineTM 2000. The mRNA expression of miR-187* and BMI-1 in HCT116 cell line was measured by real-time RT-PCR. Cell growth and cell cycle were assayed by MTS method and flow cytometry. RESULTS:miR-187* was found to be differentially expressed between colorectal adenocarcinoma and normal tissues. The expression of miR-187* in 8 colon cancer cell lines was down-regulated, while BMI-1 mRNA was up-regulated. Compared with blank control group, miR-187* expression was remarkably increased after transfection with miR-187* mimics, and ectopic expression of miR-187* significantly inhibited the mRNA expression of BMI-1. The cell growth was inhibited in miR-187* mimics group, and proliferating cell nuclear antigen (PCNA) mRNA expression was decreased. The cells at G2/M phase in miR-187* mimics group were significantly increased. CONCLUSION: miR-187* is down-regulated in human colon cancer cell lines. Up-regulation of miR-187* not only inhibits the proliferation but also influence the cell cycle of HCT116 cells, which might act as a tumor suppressor in colorectal cancer by inhibiting the expression of BMI-1.     

Modeling individual leaf area of ginger (Zingiber officinale Roscoe) using leaf length and width

Leaf area estimation is an important biometrical observation one has to do for comparing plant growth in field and pot experiments. In this study, a leaf area estimation model was developed for ginger (Zingiber officinale Roscoe), using linear measurements of leaf length (L) and maximum width (W). Leaves from five ginger varieties (Varada, Rejatha, Mahima, Maran and Himachal) were used to develop the model in 2006–2007. The actual leaf area (LA) was measured with a leaf area meter (LI-3100, LI-COR, Lincoln, NE, USA) and taken as reference LA. The linear measurements were used to build linear (LA = a + b × L × W) and power models (LA = α × (L × W)^β) for each variety, as the modeling among variety were not different from each other, data for all five varieties have been pooled and compared with earlier models by graphical procedures and statistical criteria such as Mean Square Error (MSE), Root Mean Square Error (RMSE) and Chi-square (χ²). The selected model was validated during 2007–2008. The validation data set was used to produce a validation model for each variety by re-estimating the model parameters to develop the estimation model and the models were compared for consistency. The predicted LA (PLA) was compared with observed LA (OLA) by graphical procedures and lack of agreement was evaluated by calculating the relative bias, estimated by the mean of differences (d) and the standard deviation (SD) of the differences. Normality test was carried out by Spearman's rank correlation coefficient (r_s) and residuals were normally distributed. Finally, the proposed model for leaf area estimation of ginger is LA = −0.0146 + 0.6621 × L × W, R² = 0.997. This model can be reliably used for estimating leaf area of ginger non-destructively. The same equation can be extrapolated to all varieties and land races of ginger as it is vegetatively propagated crop with narrow genetic variability.